Uses of il-12 and the il-12 receptor positive cell in tissue repair and regeneration

ABSTRACT

The present application relates to stem cells isolated from various sources within the body of a patient or of a healthy donor and identified by the presence of the interleukin 12 (IL-12) receptor. The present application also provides methods for making and for using the stem cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/824,224, which is the National Phase of International PatentApplication No. PCT/US2011/053450, filed Sep. 27, 2011, and published asWO 2012/050829, which claims priority from U.S. Provisional PatentApplication No. 61/387,419, filed Sep. 28, 2010, U.S. Provisional PatentApplication No. 61/405,584, filed Oct. 21, 2010, U.S. Provisional PatentApplication No. 61/409,407, filed Nov. 2, 2010, and U.S. ProvisionalPatent Application No. 61/477,130, filed Apr. 19, 2011, each of whichare incorporated herein by reference in their entirety, including allfigures and tables.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support under contractnumber BAA-BARDA-08-08 awarded by the Biomedical Advanced Research andDevelopment Authority, with the Department of Health and Human Services.The Government has certain rights in the invention.

BACKGROUND

Hematopoiesis is sustained by a rare population of hematopoietic stemcells (HSCs) capable of self-renewal and differentiation into multiplehematopoietic lineages. These HSCs include a long-term repopulating(LTR) subset that is capable of complete hematopoietic regeneration. Theability to maintain or expand the LTR population of hematopoietic stemcells in vitro and in vivo without inducing their differentiation iscrucial for clinical applications, such as gene therapy and theexpansion of stem cells and progenitor cells for transplantation.

During the last decade, considerable progress has been made towards theisolation and characterization of primitive hematopoietic cellpopulations in mice and humans From this body of work, severalhematopoietic stem cell identifiers, i.e., cell-surface markers found onhematopoietic stem cells, have been delineated which have proven to behighly useful for the identification and isolation of hematopoietic stemcells. Novel identifiers of hematopoietic, long-term repopulating cellsremain of interest, as the identification of novel stem cell markers mayprovide access to previously unknown subsets of rare hematopoietic stemcells.

Despite the number of cell surface markers that have been delineated todate, the identification and uses of rare populations of hematopoieticstem cells capable of tissue repair and regeneration remains unchartedterritory. Rare stem cell populations that are related to lethalradiation survival in animal models might prove to be useful in thetherapeutic areas of repair and regeneration of hematopoiesis and alsoother tissues types within the body, such as brain tissue, lung tissue,kidney tissue, pancreatic tissue, liver tissue, or cardiac tissue andthe like. Thus, the identification of novel stem cell markers that candelineate a rare population of stem cells useful in hematopoietic tissuerepair and regeneration, as well as general tissue repair andregeneration, are still needed in the areas of regenerative medicine.

SUMMARY OF INVENTION

In one embodiment, the present invention relates to a cell populationcomprising a substantially homogenous population of cells that expressesthe IL-12 receptor and the cell marker CD34 and that has been exposed toexogenous IL-12 ligand. In another embodiment, the cell population is ofhuman origin. For example, the cells can have been isolated before beingexposed to exogenous IL-12 ligand. Alternatively, the cells can havebeen exposed to exogenous IL-12 ligand while in a subject, and thenisolated. Also encompassed by the invention is a population of bloodcells differentiated from a cell population of the invention.

In one embodiment, the cell population of the invention can furtherexpress the marker CDCP1, the marker c-kit, the marker KDR, the markerFlt3, the marker SLAM, the marker CD133, the marker IFNGR (Interferon-γreceptor), the absence of CD34 as a marker, or any combination thereof.

In yet another embodiment, the stem cell present in the cell populationof the invention does not express at least one or more majorhistocompatibility (MHC) class I and class II molecules.

In one embodiment, the cell population of the invention undergoesexpansion in the presence of IL-12 heterodimer ligand. In anotherembodiment, the cell population of the invention comprises long-termrepopulating (LTR) hematopoietic stem cells. In yet another embodiment,the cell population of the invention is radioresistant.

In one embodiment, the source of the the cell population of theinvention is bone marrow. In another embodiment, the source of the cellpopulation of the invention is peripheral blood. In yet anotherembodiment, the source of the cell population of the invention is thespleen. In another embodiment, the source of the cell population of theinvention is blood from an umbilical cord.

In one embodiment, for the cell population of the invention the IL-12receptor comprises the beta 2 subunit of the IL-12 receptor.

The invention also encompasses methods for generating a cellulartransplant for repair of cells and tissue. Such a method can comprise,for example, (a) isolating a cell population that expresses the IL-12receptor and CD34; and (b) exposing the cell population to exogenousIL-12. The method can further comprise a step where the population ofcells expressing the IL-12 receptor is isolated using an antibody thatbinds the beta 2 subunit of the IL-12 receptor.

The invention further encompasses methods for repairing cells and tissuein a subject. Such a method can comprise (a) isolating a cell populationthat expresses the IL-12 receptor; (b) exposing the cell population toexogenous IL-12.; and (c) administering the cell population to asubject. In such a method, the cell population can be isolated using anantibody that binds the beta 2 subunit of the IL-12 receptor. Inaddition, the method can further comprise administering IL-12 ligand tothe subject following administration of the cell population. In yetanother embodiment, the method encompasses treating a subject withdiabetes. Alternatively or in addition, the subject can have one or moreblood cell counts that are below normal range due to the effects ofradiation therapy or chemotherapy. In yet another embodiment, the methodencompasses administering the cell population to an organ selected fromthe group consisting of kidney, liver, lung, spleen, pancreas, andcardiac tissue.

The cell population to be administered to a subject in the methods ofthe invention can include one or more pharmaceutically acceptableexcipients.

The invention further encompasses cell populations derived fromdifferentiated tissues that express the IL-12 receptor. For example,cell populations derived from neuronal tissue comprising a substantiallyhomogenous population of cells that expresses the IL-12 receptor andthat has been exposed to exogenous IL-12 ligand. The cell populationsmay also be derived from kidney tissue, uterine tissue, stomach tissue,intestinal tissue, appendix tissue, or testis tissue.

The invention further encompasses methods of regenerating various typesof tissue of a subject in vivo following administration of IL-12 ligandto the subject, wherein the IL-12 ligand binds to the IL-12 receptor ona population of cells in the tissue to yield an increase in the numberIL-12 receptor positive stem cells in that tissue. In some embodiments,the type of tissue may be neuronal tissue, kidney tissue, uterinetissue, stomach tissue, or intestinal tissue. In some eIn someembodiments, the tissue is neuronal tissue and a route IL-12administration is selected from the group consisting of epidural,peridural, intracerebral, intrathecal, and intracerebroventricular. Insome embodiments, the tissue is kidney tissue. In some embodiments, thetissue is uterine tissue. In some embodiments, the administration ofIL-12 is intrauterine. In some embodiments, the tissue is stomachtissue. In some embodiments, the tissue is intestinal tissue. In someembodiments, the IL-12 administration is enteral. In some of theforegoing embodiments, the route of IL-12 administration can besubcutaneous, intravenous, intraarterial, intramuscular orintraperitoneal.

The foregoing general description and following brief description of thedrawings and the detailed description are exemplary and explanatory andare intended to provide further explanation of the invention as claimed.Other objects, advantages, and novel features will be readily apparentto those skilled in the art from the following detailed description ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Kaplan-Meier plot of survival data for irradiated micereceiving a low dose of IL-12, as described in Example 1. Mice weretreated intravenously with rMuIL-12 (100 ng/mouse, about 5 μg/kg) at 24hours before (▴) or 1 hour after (Δ) lethal irradiation (10 Gy). Controlgroup mice (□) were injected with the same volume of phosphate bufferedsaline. The p value was calculated using Log Rank Test (IL-12 treatmentversus control, p<0.001; IL-12-treated before IR versus after IR,p<0.05).

FIGS. 2A-2F shows graphs of peripheral blood cell counts from irradiatedmice receiving IL-12, as described in Example 4. FIGS. 2A-2C are formice receiving lethal irradiation (10 Gy), FIGS. 2D-2F are for micereceiving sublethal irradiation (5 Gy). The Y axis indicates thepercentage change of the cell count for each blood cell subtype afterradiation in relative to its baseline blood cell count before radiation.(*; p<0.05; **: p<0.01, Error bars represents S.E.M.). Arrow indicatesthe time of radiation. 0 day means the day of radiation. Bg: background.

FIG. 3 shows photomicrographs of femurs sectioned from animals treatedwith and without IL-12, as described in Example 4. The femurs were fixedand stained for hematoxylin and eosin. Arrows at 1 day point tohemorrhage. Arrows at 12 days point to mononuclear colonies. 14 daysshows the overall increase in regeneration after IL-12 treatment 200×magnification. d: days post radiation.

FIGS. 4A-4F show graphs of various assays on bone marrow cells fromLy5.2 mice treated with IL-12 twenty four hours before irradiation, asdescribed in Example 5. FIG. 4A shows a bar graph of Ly5.1 mousesurvival following transplantation of Ly5.2 bone marrow cells in arepopulation assay, while FIG. 4B shows a bar graph for results of aCFU-S₁₂ assay of spleens from mice that received transplanted cells.FIG. 4C shows a graph of data from a colony-forming cells assay on bonemarrow cells from Ly5.1 mice treated with IL-12 twenty four hours beforeirradiation. FIG. 4D shows a graph representing cell counts of Ly5.2cells that have repopulated a Ly5.1 animal receiving an Ly5.2 bonemarrow transplant. FIG. 4E shows a representative plot of flow cytometrydata for a subpopulation of myeloid cells in a population oftransplanted bone marrow cells. FIG. 4F shows a representative plot offlow cytometry data for a subpopulation of myeloid cells in a populationof transplanted bone marrow cells.

FIG. 5 shows a schematic of MSC regulation via the IL-12 ligand/IL-12receptor system as controlled by dendritic cells (DC), as described inExample 7. IL-12 produced by activation of DC, or exogenous IL-12, cancontrol the fate of HSC. HSC are characterized by an increased cellcycle quiescence compared to other cells. They can remain in quiescence,enter cell cycle to undergo symmetric or asymmetric division, or undergoapoptotic death. Recent reports indicate that bone marrow cells areregulated by DC (91). The “status” of IL-12, i.e., the concentration ofIL-12, in circulation affects equilibria involved in the production ofLTR HSC (symmetrical cell division producing two LTR) or STR HSC(symmetrical cell division producing two STR). Also the “status” ofIL-12 can affect equilibria leading to the quiescent state or apoptosis.When the level of IL-12 is “high”, largely symmetrical cell divisionwill take place, but whether LTR or STR are mainly produced depends onother signals. When the status of IL-12 is “low”, the equilibria arepushed toward asymmetrical cell division, leading to homeostasis, orquiescence, again depending on other signals.

FIGS. 6A and 6B are photomicrographs of IL-12Rβ2 expression in human andrhesus intestinal crypts. FIG. 6A shows representative IL-12Rβ2expression on intestinal stem cells (circled). FIG. 6B shows IL-12Rβ2expression on intestinal stem cells (circled) and paneth cells (arrows).

FIGS. 7A and 7B are photomicrographs of expression of IL-23Rβ2 in normalsalivary gland of a human. IL-12Rβ2 is predominately expressed insalivary secretory acini (AC) of the salivary gland. IL-12Rβ2 expressionis also represented in striated excretory ducts (ED) of the salivarygland.

FIG. 8 is a series of photomicrographs of mouse femoral bone marrowstained for IL12Rβ2, as described in Example 8. The mice were subjectedto total body irradiation (8 Gy) and received either vehicle or rMuIL-12at various times post-irradiation.

FIGS. 9A-9B is a series of photomicrographs of femoral bone marrowstained for IL-12Rβ2 and Sca-1, or IL-23Rβ2 and osteocalcin, asdescribed in Example 8. FIG. 9A shows tissue sections obtained 30 daysafter total body irradiation; FIG. 9B shows tissue sections obtained 12days after total body irradiation. Magnification is 100×.

FIG. 10A is a photomicrograph of mouse jejunal crypts stained forIL-12Rβ2, as described in Example 8. FIG. 10B is a series ofphotomicrographs of jejunal crypts from mice treated with rMuIL-12,either without irradiation (top panels) or with irradiation (8.6 Gy;bottom panels).

FIG. 11A is a series of photomicrographs of femoral bone marrow fromnon-human primate (NHP) or human, stained for IL-12Rβ2, as described inExample 9. FIG. 11B is a series of photomicrographs of jejunum/iliumfrom non-human primate (NHP) or human, stained for IL-12Rβ2.

FIG. 12A is a Kaplan-Meier plot of survival data for individual dosinggroups of monkeys after receiving IL-12 either 24 hours following totalbody irradiation, or at both 24 hours and 7 days following total bodyirradiation, as described in Example 10. FIG. 12B is a Kaplan-Meier plotof survival data for pooled data from FIG. 12A.

FIG. 13A is a graph of leukocyte counts from monkeys followingirradiation and treatment with IL-12, as described in Example 11. FIG.13B is a graph of platelet counts from monkeys following irradiation andtreatment with IL-12 as described in Example 11.

FIG. 14A is a plot of flow cytometry data for lineage marker-depletedhuman bone marrow cells labeled for IL-12Rβ2 and CD34, as described inExample 12. The quadrants were set based on unstained and isotypecontrols. R14=IL-12Rβ2⁺CD34⁻, R15=IL-12Rβ2⁺CD34⁺, R17=IL-12Rβ2⁺CD34⁺.FIG. 14B is a plot of flow cytometry data for lineage marker-depletedhuman bone marrow cells labeled for IL-12Rβ2 and CD34. The quadrantswere set based on unstained and isotype controls. R2=IL-12Rβ2⁺CD34⁻,R3=IL-12Rβ2⁺CD34⁺, R5=IL-23Rβ2⁻CD34⁺. FIG. 14C is a photomicrograph ofCD34⁺ cells stained for IL-23Rβ2.

FIG. 15A is a bar graph showing percentages of human lineagemarker-depleted, IL-12Rβ2-expressing cell subsets that co-express withthe hematopoietic stem cell (HSC) markers CD34, ckit, KDR, CD133, Flt3,SLAM and CDCP1, as described in Example 12. FIG. 15B is a bar graphshowing percentages of human CD34⁺, IL-12Rβ2-expressing cell subsetsthat co-express with HSC markers CD34, ckit, KDR, CD133, Flt3, SLAM andCDCP1.

FIG. 16 shows plots of flow cytometry data for expression ofintracellular IL-12Rβ2 in human bone marrow CD34⁺ cells cultured inmedia alone (left panel) or in the presence of 10 pM IL-12 (rightpanel), as described in Example 13.

FIG. 17A shows plots of flow cytometry data for expression of cellsurface IL-12Rβ2 and CD34 on human bone marrow Lin⁻ cells, as describedin Example 13. The quadrants were set based on unstained and isotypecontrols. R14=IL-12Rβ2⁺CD34⁻, R15=IL-12Rβ2⁺CD34⁺. FIG. 17B shows a bargraph of data for amount of CFU-GEMM colony formation in IL-12Rβ2⁺CD34⁻and IL-12Rβ2⁺CD34⁺ cultures with and without IL-12 stimulation on Day 8of culture, as described in Example 13.

FIGS. 18A-18B shows plots of flow cytometry data (forward scatter andside scatter) for human lineage marker-depleted stem cells, as describedin Example 13. FIG. 18A and 18B are from two different donor subjects.

FIGS. 19A-19D shows survival data and cell count data from mice treatedeither with IL-12 or bone marrow transplant twenty four hours followinglethal irradiation, as described in Example 15. FIG. 19A is aKaplan-Meier survival plot of control, IL-12-treated, and bone marrowtransplant treated mice. FIG. 19B-D are graphs showing blood cell countsfrom the treated and control groups at various time points followingirradiation. The dashed line represents the cutoff point for a cellcount in a healthy mouse. FIG. 19B shows the counts for neutrophils,FIG. 19C shows the counts for red blood cells, and FIG. 19D shows thecounts for platelets.

FIGS. 20A and 20B show photomicrographs of bone marrow from micefollowing lethal irradiation as described in Example 15 (FIG. 20Acontrol; FIG. 20B, treated).

FIG. 21A shows a Kaplan-Meier survival plot of mice that receivedvarious doses of radiation, followed by a single dose of IL-12 twentyfour hours following irradiation as described in Example 16.

FIG. 21B shows a Kaplan-Meier survival plot of mice that received asingle dose of irradiation followed by a single dose of IL-12 at varioustimes following irradiation as described in Example 17.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the identification, isolation andcharacterization of rare, survival-related stem cells delineated by apreviously unknown stem cell marker and its associated ligand. The novelstem cell marker of the invention is the Interleukin-12 receptor(IL-12R) and the ligand is Interleukin-12 (IL-12). The novel stem cellof the invention, which is marked by the IL-12 receptor, with or withoutthe use of its associated ligand can be used, for example, for repairand regeneration of various tissue types, including blood, bone marrow,as well as cardiac, brain, pancreatic, renal or liver tissue or thelike.

The ligand Interleukin-12 (IL-12) is well-known for its immunoregulatoryproperties working at the level of differentiated, mature hematopoieticcells, such as natural killer cells and other T-cells. A significantbody of work has demonstrated that IL-I 2 is a potent immuno-modulatorwith significant anti-tumorigenic and anti-angiogenic properties. IL-12,however, is generally not known to play a role in hematopoiesis at thelevel of hematopoietic stem cells (HSC). Based on the fact thatInterleukin-12 (IL-12) can uniquely confer survival to lethallyirradiated mice, as shown in FIG. 1, a hypothesis was generated whichyielded the present invention. In accordance with the invention, aresponsive IL-12 receptor positive stem cell (IL-12R⁺) exists in thebody that can be activated by the IL-12 ligand. Further, the IL-12R⁺stem cell of the present invention possesses significant survivalrelated properties that are involved in the repair and regeneration oftissue.

Herein is provided evidence that the IL-12R⁺ stem cell is involved inhematopoietic stem cell survival and expansion. Evidence is providedherein that merely one, low dose of IL-12 to mice, either shortly beforeor after the administration of a lethal dose of radiation, produces thefollowing properties:

-   -   IL-12 confers significant survival to lethally irradiated mice        by generating multilineage, hematopoietic recovery following        lethal radiation. No single cytokine can confer survival to        lethally irradiated animals when administered as one parenteral        dose that can be administered either before or after a lethal        dose of radiation.    -   IL-12 administration leads to an increase in bone marrow        cellularity and expansion of certain hematopoietic/progenitor        stem cells    -   Donor hematopoietic cells derived from mice rescued from the        deleterious effects of lethal radiation via IL-12 administration        are capable of repopulating secondarily lethally irradiated        murine recipients.

These novel and unexpected findings suggest that the IL-12 ligand actingon an IL-12R⁺ stem cell have a significant and previously unrecognizedrole in hematopoietic stem cell survival and in vivo stem cellexpansion. Thus, the present invention includes isolating and utilizingthe IL-12-responsive hematopoietic “survival” stem cell for use inhematopoietic stem cell applications, such as ex vivo hematopoietic stemcell expansion and differentiation, as well as repair and regenerationof various tissues, such as bone marrow, cardiac, lung, brain,pancreatic, kidney or liver tissue or the like. Thus, the inventionyields a rare subset of hematopoietic stem cells with highproliferative, repopulating, repair and regeneration potential. Thisprimitive and survival-conferring stem cell population may also beuseful in ex-vivo hematopoietic stem cell expansion and differentiationinto mature blood cells, including white blood cells, red blood cellsand platelets.

The isolation of the human, IL-12 responsive, survival-conferring stemcells, either with or without the use of the IL-12 ligand, can be usedcommercially, for example, (1) to generate highly reparative andregenerative, cellular transplants, and (2) to generate in vivo and exvivo expanded hematopoietic stem cell populations, includingdifferentiated blood cells produced from the expanded HSC, and variousother applications as described herein. In this regard, a correspondingantibody IL-12R can be generated to the human IL-12R⁺ stem cell.

A. Definitions

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent depending uponthe context in which it is used. If there are uses of the term which arenot clear to persons of ordinary skill in the art given the context inwhich it is used, “about” will mean up to plus or minus 10% of theparticular term.

As used in this disclosure, except where the context requires otherwise,the term “comprise” and variations of the term, such as “comprising,”“comprises” and “comprised” are not intended to exclude other additives,components, integers or steps.

The term “expansion” is defined herein as an increase in the number ofcells in a cell population.

The term “hematopoietic stem cell” or “HSC” as used herein refers to apopulation of stem cells derived from a population of blood cells thatcan differentiate into multiple types of cells.

The term “IL-12 heterodimer ligand” is defined herein as aheterodimeric, soluble protein (also known as IL-12R P70) thatspecifically hinds to and activates the IL-12 receptor where the IL-12receptor is a membrane-bound cellular protein. The IL-12 heterodimerligand has a p35 subunit (SEQ ID NO: 1; GenBank Accession NumberNP_(—)000873) and a p40 subunit (SEQ ID NO: 2; GenBank Accession NumberNP_(—)002178) which, after removal of signal sequence peptides, combineto form a heterodimer. In accordance with the present invention, theIL-12 heterodimer ligand may or may not be glycosylated. Also in thepresent invention the IL-12 heterodimer ligand binds to the IL-12receptor expressed on a stem cell. The preferred amino acid sequence forthe IL-12 heterodimer ligand is as follows, but variant forms containingpolymorphisms are in accordance with the present invention.

IL-12 Ligand p35 Subunit [SEQ ID NO: 1]: 1rnlpvatpdp gmfpclhhsq nllraysnml qkarqtlefy pctseeidhe 61ditkdktstv eaclpleltk nesclnsret sfitngscla srktsfmmal 111clssiyedlk myqvefktmn akllmdpkrq ifldqnmlav idelmqalnf 161nsetvpqkss leepdfyktk iklcillhaf riravtidrv msylnasIL-12 Ligand p40 Subunit [SEQ ID NO: 2]: 1iwelkkdvyv veldwypdap gemvvltcdt peedgitwtl dqssevlgsg 61ktltiqvkef gdagqytchk ggevlshsll llhkkedgiw stdilkdqke 111pknktflrce aknysgrftc wwlttistdl tfsvkssrgs sdpqgvtcga 161atlsaervrg dnkeyeysve cqedsacpaa eeslpievmv davhklkyen 211ytssffirdi ikpdppknlq lkplknsrqv evsweypdtw stphsyfslt 261fcvqvqgksk rekkdrvftd ktsatvicrk nasisvraqd ryyssswsew 311 asvpcs

The term “IL-12 homodimer ligand” is defined herein as a soluble,dimeric protein comprising two p40 subunits (see SEQ ID NO: 2) to formthe homodimer. The IL-12 homodimer may or may not be glycosylated. TheIL-12 homodimer also binds to the IL-12 receptor. In the presentinvention, the IL-12 homodimer can bind to and further activate ormodify the activity of the IL-12 ligand/IL-12 receptor complex bound tothe surface of the IL-12 stem cell. In the invention, the IL-12homodimer may be administered to a patient or donor exogenously.Although sequence variation may be tolerable to the limits of thepresent invention, the preferred sequence for each monomer of the IL-12homodimer is that of SEQ ID NO: 2.

The term “IL-12 monomer ligand” is defined as protein comprising the p40subunit of the IL-12 ligand (SEQ ID NO: 2). The IL-12 monomer may or maynot be glycosylated, The IL-12 monomer also binds to the IL-12 receptor.In the invention, the IL-12 monomer may be administered to a patient ordonor exogenously. In the present invention, the IL-12 monomer can bindto and further activate or modify the activity of the IL-12 ligand/IL-12receptor complex bound to the surface of the IL-12 stem cell.

The term “IL-12 receptor” is defined herein as a heterodimeric,membrane-bound receptor for the IL-12 ligand. The IL-12 receptorheterodimer subunits are beta 1 (β1) and beta 2 (β2). In accordance withthe present invention, the IL-12 receptor may also bind the IL-12homodimer and the IL-12 monomer, as defined herein, to form a multimercomplex comprising the IL-12 ligand/IL-12 receptor pair and thehomodimer and/or the monomer. In the present invention, the multimercomplex would further activate the IL-12 ligand/IL-12 receptor pair ormay modify the activity of the ligand/receptor pair. In accordance withthe present invention, the IL-12 receptor protein is defined to be inits endogenous state as isolated from the IL-12 selected stem cell takenfrom a donor or a patient. As such, the IL-12 receptor may containpolymorphisms distinct from the canonical amino acid sequence of the β1(SEQ ID NO: 3; from GenBank Accession Number NP_(—)005526) and β2subunits (SEQ ID NO: 4; from GenBank Accession Number NP_(—)001550), asdescribed below:

IL-12 receptor (beta 1) [SEO 1D NO: 3]: 1meplvtwvvp llflfllsrq gaacrtsecc fqdppypdad sgsasgprdl rcyrissdry 61ecswqyegpt agvshflrcc lssgrccyfa agsatrlqfs dqagvsvlyt vtlwveswar 121nqtekspevt lqlynsvkye pplgdikvsk lagqlrmewe tpdnqvgaev qfrhrtpssp 181wklgdcgpqd ddtesclcpl emnvaqefql rrrqlgsqgs swskwsspvc vppenppqpq 241vrfsveqlgq dgrrrltlke gptqlelpeg cqglapgtev tyrlqlhmls cpckakatrt 301lhlgkmpyls gaaynvavis snqfgpglnq twhipadtht epvalnisvg tngttmywpa 361raqsmtycie wqpvgqdggl atcsltapqd pdpagmatys wsresgamgq ekcyyitifa 421sahpekltlw stvlstyhfg gnasaagtph hvsvknhsld svsvdwapsl lstcpgvlke 481yvvrcrdeds kqvsehpvqp tetgvtlsgl ragvaytvqv radtawlrgv wsqpqrfsie 541vqvsdwliff aslgsflsil lvgvlgylgl nraarhlcpp lptpcassai efpggketwq 601winpvdfqee aslqealvve mswdkgerte plektelpeg apelaldtel sledgdrcka 661 kmIL-12 receptor (beta 2) [SEQ ID NO: 4]: 1mahtfrgcsl afmfiitwll ikakidackr gdvtvkpshv illgstvnit cslkprqgcf 61hysrrnklil ykfdrrinfh hghslnsqvt glplgttlfv cklacinsde iqicgaeifv 121gvapeqpqnl sciqkgeqgt vactwergrd thlyteytlq lsgpknltwq kqckdiycdy 181ldfginltpe spesnftakv tavnslgsss slpstftfld ivrplppwdi rikfqkasvs 241rctlywrdeg lvllnrlryr psnsrlwnmv nvtkakgrhd lldlkpftey efqissklhl 301ykgswsdwse slraqtpeee ptgmldvwym krhidysrqq islfwknlsv seargkilhy 361qvtlqeltgg kamtqnitgh tswttviprt gnwavavsaa nskgsslptr inimnlceag 421llaprqvsan segmdnilvt wqpprkdpsa vqeyvvewre lhpggdtqvp lnwlrsrpyn 481vsalisenik syicyeirvy alsgdqggcs silgnskhka plsgphinai teekgsilis 541wnsipvqeqm gcllhyriyw kerdsnsqpq lceipyrvsq nshpinslqp rvtyvlwmta 601ltaagesshg nerefclqgk anwmafvaps iciaiimvgi fsthyfqqkv fvllaalrpq 661wcsreipdpa nstcakkypi aeektqlpld rllidwptpe dpeplvisev lhqvtpvfrh 721ppcsnwpqre kgiqghqase kdmmhsassp pppralqaes rqlvdlykvl esrgsdpkpe 781npacpwtvlp agdlpthdgy lpsniddlps heapladsle elepqhisls vfpssslhpl 841tfscgdkltl dqlkmrcdsl ml

The term “IL-12 antibody” is defined herein as antibody that binds toone or more epitopes to the 11-12 beta 2 receptor subunit or the complexof the IL-12 beta 1/IL-12 beta 2 subunits.

The term “IL-12 stem cell” is defined herein as an isolated cell derivedfrom a population of blood cells that does not express lineage markers,i.e., an immature cell, and expresses the IL-12 receptor. The IL-12 stemcell will comprise other membrane-bound surface markers. The preferredsource of the isolated IL-12 stem cell is human bone marrow, but otherblood sources are suitable to the present invention. These are humancells derived from peripheral blood, blood cells derived from spleen,and cord blood. In accordance with the present invention, the IL-12 stemcell is activated by the IL-12 ligand. Also in accordance with theinvention, activation by the IL-12 ligand can be further modified by thepresence of the IL-12 homodimer or monomer interacting with the IL-12ligand/IL-12 receptor pair.

The term “IL-12 stem cell transplant” is defined herein as a populationof cells comprising the IL-12 stem cell as defined herein. In accordancewith the invention, this population of cells may comprise other cells ascarrier cells. The choice of carrier cells will depend on the targettissue that will receive the transplant. For example, if the targettissue is the bone marrow compartment, then the carrier cells willgenerally be a population of blood cells. If the target tissue is otherthan bone marrow, the carrier cells may be derived from the targetedrecipient organ or may be blood cells. In the invention the IL-12 stemcell is derived from a population of immature blood cells.

The term “lineage deficient” or “Lin⁻” describes cells that lack certainmarkers, indicating that the cells are not committed to producing aparticular cell type lineage. For example, hematopoietic stem cells arelineage deficient when they lack the markers CD3e, CD4, CD5, CD8b, CD8a,B220, CD11b, Gr1 and Ter.

As used herein, “pluripotent stem cell” means a stem cell that candifferentiated into two or more differentiated cell types. For example,differentiated cell types can include blood cells, neural cells,endothelial cells, cardiac cells, pancreatic cells, kidney cells, livercells, spleen cells and lung cells.

The term “population of cells” is defined herein as a collection ofcells comprising the IL-12 stem cell as defined herein.

The phrase “repair and regeneration of diseased organs” is definedherein as some measurable or quantifiable increase in the health of thetargeted recipient organ. Repair and regeneration of diseased organs caninvolve, but is not limited to, transdifferentiation of the IL-12 stemcell or carrier cells into cells related to the targeted recipient organwhere the targeted recipient organ is other than bone marrow tissue orblood tissue.

The term “short-term repopulating hematopoietic stem cells” (STR HSC) isdefined herein as hematopoietic stem cells that are capable supportinghematopoiesis for no more than 15 weeks. STR HSC can have a cell markerprofile of: CD34⁺, SCA-1⁺, Thy1.1^(−/lo), C-kit⁺, Lin⁻, CD135⁻,Slamf1/CD150⁺, Mac-1 (CD11b)^(lo).

The term “long term repopulating hematopoietic stem cells” (LTR HSC) isdefined herein as hematopoietic stem cells that are capable ofsupporting hematopoiesis in an animal for 6 months or longer. LTR HSCcan have different cell marker profiles depending on the animal speciesfrom which they are derived. For example, mouse LTR HSC have a cellmarker profile of: CD34⁻, SCA-1⁺, C-kit⁺, Lin⁻, Slamf1/CD150⁺, CD135⁻.

As used herein “a substantially homogenous cell population” refers to apopulation or sample of cells which contain a majority (i.e., at least50%) of cells having the trait(s) of interest. In preferred embodiments,substantially homogenous populations contain at least 60%, at least 70%,at least 80%, at least 90% or more of the cells having the trait(s) ofinterest.

The term “therapeutically effective amount or dose” is defined herein asa dose of a substance that produces effects for which it isadministered. For example, a dose of IL-12 sufficient for increasingsurvival and/or preserving bone marrow function, and/or promotinghematopoietic recovery or restoration in a subject followingmyeloablation, radiation therapy, and/or chemotherapy. The exact dose ofIL-12 will depend on the purpose of the treatment, the timing ofadministration of IL-12, certain characteristics of the subject to betreated, the total amount or timing of myeloablation, radiation therapy,and/or chemotherapy, and is ascertainable by one skilled in the artusing known techniques (see, e.g., Lieberman, Pharmaceutical DosageForms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20^(th) Edition,2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

Generally, a dose of a therapeutic agent, according to the methods andcompositions of the present invention, can be expressed in terms of thetotal amount of drug to be administered, (i.e. ng, g, or mg).Preferably, the dose can be expressed as a ratio of drug to beadministered to weight or surface area of subject receiving theadministration (i.e., ng/kg, g/kg, ng,/m², or g/m²). When referring to adose in terms of the mass to be administered per mass of subject (i.e.,ng/kg), it will be understood that doses are not equivalent betweendifferent animals, and thus conversion factors will need to be used toensure that one animal receives the same dose equivalent as anotheranimal. Suitable factors for the conversion of a mouse “dose equivalent”to a “dose equivalent” of a different animal are given in Table 1 below.

TABLE 1 Conversion Factors and Equivalent Doses for Several AnimalsWeight Total Dose Dose Dose Conversion Species (kg) (ng/kg) (ng/kg)(ng/m²) Factor Human 65 25655.82 394.7 15,000 0.0794 Mouse 0.02 99.474973.44 15,000 1.0000 Hamster 0.03 130.2 4339.87 15,000 0.8726 Rat 0.15381.12 2540.8 15,000 0.5109 Guinea Pig 1.00 1335 1335 15,000 0.2684Rabbit 2.0 2381.1 1190.65 15,000 0.2394 Cat 2.5 2956.44 1182.57 15,0000.2376 Monkey 3.0 3681.75 1227.25 15,000 0.2468 Dog 8.0 6720 840 15,0000.1689Thus, in one embodiment, doses are given in terms of mass to surfacearea (i.e., ng/m² or g/m²), which are equivalent for all animals. Thefollowing basic conversion factors can be used to convert ng/kg tong/m²: mouse=3.0, hamster=4.1, rat=6.0, guinea pig=7.7, human=38.0(Cancer Chemother Repts., 50(40):219(1966)).

The term “tissue committed stem cell” or “TCSC” is defined herein as astem cell that is not fully differentiated, but can only differentiateinto cells that make up a certain type of tissue, for example, livercells or blood cells.

B. Stem Cells and Stem Cell Factors

Stem cells have been shown to possess vast therapeutic potential. (9)Thus, it is anticipated that advances in stem cells as therapeutics willhave a tremendous impact on the clinical practice of medicine. Thehematopoietic stem cell, the common ancestor of all types of bloodcells, is the best-characterized stem cell in the body and the only stemcell that is clinically applied in the treatment of diseases thatinclude malignancies such as leukemia, lymphomas, myeloma, pediatricneuroblastoma and sarcomas, as well as congenital immunodeficiencies andbone marrow failure (e.g., aplastic anemia).

New techniques involving multicolor cell sorting can enable thepurification of hematopoietic stem cells and their downstreamprogenitors, such as common lymphoid progenitors and common myeloidprogenitors (10,11). Recent genetic approaches, including gene chiptechnology, are useful in elucidating the gene expression profile ofhematopoietic stem cells (12-14).

Another important aspect related to recent advances in hematopoieticstem cells as therapeutics is the discovery of hematopoietic stem cellfactors, and their cognate receptors, that can be used to identify andregulate (activate) stem cells in vivo and in vitro. Such advances arepropelling the therapeutic use of hematopoietic stem cells closer tofruition as treatment modalities for a host of diseases and disorders.Further, the availability of novel stem cell factors for themanipulation of hematopoietic stem cells may be the necessary link thatrenders populations of rare stem cells accessible to physicians andscientists for use in medicine and research.

The present invention is based on the discovery that Interleukin-12(IL-12) can uniquely confer survival to lethally irradiated mice at lowdoses. These studies provided inferential evidence that IL-12 may beinvolved in hematopoietic stem cell survival and expansion. Just one lowdose of IL-12 to mice, either shortly before or after the administrationof a lethal dose or radiation, confers survival and multilineage,hematopoietic recovery to the lethally irradiated subject. Further,these effects originate in the protection and/or expansion of LTR HSC asevidenced by the ability of donor bone marrow cells from previouslyrescued mice to rescue a secondarily lethally irradiated recipient, aswell as other data (See FIG. 2).

These novel and unexpected findings suggest that IL-12 has a significantand previously unrecognized role in hematopoietic stem cell survival andin vivo stem cell expansion. Thus, the invention involved the isolationand utilization of the corresponding IL-12R⁺ stem cell, which hasnumerous applications in vivo and ex vivo.

C. Clinical Need for Ex Viva Expanded Hematopoietic Stem Cells

Cord blood (CB), peripheral blood (PB) or bone marrow (BM)-derivedhematopoietic stem cells provide therapeutically efficacious sources ofcells to treat a variety of hematological disorders (15,16), as well asfor use in tissue repair and regeneration. Unfortunately, the lownumbers of HSC isolated from a typical CB, PB or BM donation placeslimitations on these therapies (15,17). Effective HSC expansionrepresents an attractive solution to numerous clinical needs (18).However, this goal has remained elusive despite more than 20 years ofexperimentation in animal models and human clinical trials (19). Eventhe seemingly attainable goal of using culture-generated progenitors toshorten neutrophil and platelet recovery times in patients followingmyeloablative chemotherapy (20-22) has been generally ineffective (19).

The ability to successfully expand hematopoietic stem cells (HSC) invivo and/or ex vivo would not only have a profound impact in the waythat HSC transplantation is perceived, but could also expand the limitsof tumor cell purging and somatic cell gene therapy (23-25), as well asbe used as transplants for tissue repair and regeneration. Thedifficulties associated with inadequate numbers of HSC collected fortransplantation from autologous or allogeneic sources, such asperipheral blood and bone marrow, or allogeneic umbilical cord bloodwould be largely eliminated. Moreover, if these difficulties areeliminated, human umbilical cord blood, which has limited use in adultsbecause of an insufficient number of stem cells, might also become areliable and attractive alternative to peripheral blood or bone marrowas a source of hematopoietic progenitors (23,26) for patients withmalignant and nonmalignant conditions who lack traditional donors(27-29).

What might have been the deterrent to previous attempts at in vivoand/or ex vivo expansion of HSC? One obstacle that is solved by thepresent invention was the lack of a suitable stem cell population andactivating ligand pair. In accordance with the present invention, theIL-12 stem cell and IL-12 ligand are capable of yielding expanding stemcell populations either in vivo or ex vivo.

Also in terms of ex vivo expansion, a recent study has revealed that thegeneration of mature blood cell populations (i.e., lin⁺ cells) and theiroverall effects on culture microenvironment is the major limitation onthe expansion of HSC in vitro (30). Madlambayan et al. (30) showed thatthe direct secretion of negative regulators by culture-generated lin⁺cells, and the indirect stimulation of cells to secrete negativeregulators by culture-conditioned media, limits in vitro HSC generation.In the present invention, this same principle can be applied to in vivoexpansion of stem cells. Thus, Madlambayan et al. developed a globalculture manipulation (GCM) strategy to abrogate these effects andproduce elevated numbers of LTC-ICs (14.6-fold relative to input),migrating rapid NOD/SCID repopulating cells (12.1-fold), and long-termNOD/SCID repopulating cells (5.2-fold) (30). This GCM approach appearsto be a novel and generally useful approach with the capacity togenerate expansion of hematopoietic stem cells leading to the productionof mature blood cell populations. Thus in the present invention, thismethod, or similar methods, is used to generate ex vivo hematopoieticstem cell expansion via the utilization of isolated, IL-12 responsive,survival-conferring stem cells. The method of Madlambayan et al. isincorporated into the present application by reference in its entirety.

D. IL-12 is a Potent Immunomodulator

In all of the following description, any reference to the IL-12 ligandimplies that there is a corresponding IL-12 receptor that is involved inthe described effects.

Interleukin-12 (IL-12) is a heterodimeric pro-inflammatory cytokine thatregulates the activity of cells involved in the immune response (31). Itstimulates the production of interferon-γ (IFN-γ) from natural killer(NK) cells and T cells (32-35), favors the differentiation of T helper 1(TH 1) cells (36,37), and forms a link between innate resistance andadaptive immunity. IL-12 has also been shown to inhibit cancer growthvia its immuno-modulatory and anti-angiogenesis effects (38-42). IL-12is produced mainly by dendritic cells (DC) and phagocytes (macrophagesand neutrophils), once they are activated by encountering pathogenicbacteria, fungi or intracellular parasites (43,44).

E. The IL-12/Interferon-γ Feedback Loop and Inhibition of Hematopoiesis:

IL-12 and INF-γ are involved in a feedback loop in vivo, where theproduction of IL-12 stimulates the production of INF-γ, which, in turn,enhances the production of IL-12. In in vitro systems, it has beenreported that IL-12 can synergize with other cytokines (IL-3 and SCF) tostimulate the proliferation and differentiation of early hematopoieticprogenitors (45-47). However, in contrast to the in vitro stimulation ofearly hematopoietic progenitors, in vivo administration of exogenousIL-12 has been shown to decrease peripheral blood cell counts and bonemarrow hematopoiesis (48). Via the use of IFN-γ receptor knockout mice,Eng et al. and Car et al. demonstrated that high dosages of IL-12 didnot induce commonly seen toxicity effects, such as lymphopenia andinhibition of hematopoiesis (49,50). This observation suggests that theenhancement of bone marrow progenitors from IL-12 is balanced in vivo bythe production of INF-γ, which acts in a dominant myelo-suppressivefashion.

F. The Role of IL-12 in Hematopoietic Regeneration Following LethalRadiation

Protection of the hematopoietic stem cell (HSC) compartment from theeffects of lethal doses of radiation provides a model system to studythe biology of stem cell regeneration. Neta et al. reported that IL-12can protect bone marrow, but sensitizes the gastrointestinal (GI)system, when administrated 18 hours before radiation at a dose of 50μg/kg (51). In these studies, the IL-12 treated mice died earlier thanthe control animals due to GI sensitization. Neta et al. also reportedthat the bone marrow protective effect of IL-12 could only be obtainedif IL-12 is administered before radiation.

As described in Examples 1 and 2, a marked hematopoietic survival effecthas been observed at a dose of 5 μg/kg (100 ng/mouse), which is 10 timeslower than the dose in the Neta study, with no toxicity to the GIsystem. These results were obtained only when IL-12 was administeredduring a somewhat restricted time “window” in relation to the time ofradiation (see Example 2). At sublethal doses of radiation (e.g., 500rad), the data also indicate that administration of IL-12 attenuates thedecrease in blood cell counts, i.e., increases blood cell counts (seeExample 3). The GI toxicity observed by Neta et al. is correlated withthe higher IL-12 dose used in Neta's studies. Also the decrease in bloodcell counts observed in human Phase I and Phase II clinical trials ofIL-12 as a cancer therapy may be due to the higher IL-12 doses, as wellas repeated administration of the biologic.

L-12 appears to be unique in its ability to effect survival of lethallyirradiated mice. The magnitude of the lethal irradiation survivaleffect, the fact that survival is achieved even when IL-12 isadministered after lethal radiation, the ability of IL-12 to effectlethal irradiation survival without the use of other cytokines, as wellas the very low dose required for the lethal irradiation survivaleffect, appear to render IL-12 superior to the lethal radiation survivaleffect of other cytokines. For example, Stem Cell Factor (SCF) canrescue 80% of lethally irradiated mice only when it is given to micebefore lethal irradiation and only at a dose that is about 3 orders ofmagnitude greater than the survival dose of IL-12 used in our studies(52). Other studies report that lethal radiation rescue can beaccomplished by administering growth factors up to 2 hours postradiation, but only with a complex combination of growth factors, namelySCF, TPO, IL-3, FLT-3 ligand and SDF-1 (53-54). Even the bi-functionalFlt-3 ligand and G-GSF fusion agonist, referred to as progenipoietin-1,can achieve lethal radiation rescue only when administered 24 hoursprior to radiation and at a dose that is three orders of magnitudehigher than the dose of IL-12 used in our preliminary studies (55).

G. IL-12-Responsive Hematopoietic “Survival” Stem Cell Properties

The IL-12-responsive HSC is related to survival, and hence, thisputative subset is referred herein as the “IL-12-responsive,hematopoietic survival stem cell.” As such, this survival stem cell is avery primitive and a normally quiescent hematopoietic stem cell. (Undercertain bodily stresses, however, such as radiation injury, or otherstresses, such as chemotherapy or surgery, where the need forhematopoiesis, or other cellular functions, increase, these normallyquiescent cells are activated by signals produced from dying cells,particularly blood cells.

Recently, the heterogeneity of bone marrow stem cells has come to thefore (56). Kucia et al. propose that bone marrow (BM) may harbor, inaddition to hematopoietic stem cells (HSC), other rare versatilesubpopulations of rare tissue-committed stem cells (TCSC), and perhaps,even more primitive pluripotent stem cells (PSC). Kucia et al. furtherpropose that these primitive stem cells accumulate in bone marrow duringontogenesis where they find a permissive environment to survive. Lookingfrom this perspective, marrow potentially could contain heterogeneouspopulations of stem cells at different levels ofdifferentiation-beginning from early PSC to TCSC. These cells arereferred to by Kucia et al. as a “reserve pool of mobile cells fortissue repair” that may be released from BM into peripheral blood aftertissue injury to regenerate damaged organs. Thus, the IL-12-responsive,survival stem cell may be a TCSC or a PSC repair-related stem cell.

Moreover, the IL-12-responsive HSC subset is radioresistant.Experimental models have delineated the toxic effects of ionizingradiation on the hematopoietic stem cell compartment (57-60). Asublethal dose of only 500 cGy has been shown to eliminate 99% of thecompetent hematopoietic stem cells based upon their ability torepopulate a lethally irradiated secondary recipient (57). However, ithas been proposed that a very small fraction of hematopoietic stem cellsmay be radioresistant (60-62). Heterogeneity of stem cell populations inthe form of radioresistance has been demonstrated for intrathymic stemcells (63,61) and short term repopulating stem cells, namely CFU-S(57,60).

The IL-12-responsive HSC subset exhibits the properties of both along-term repopulating (LTR) and a short-term repopulating (STR) HSC bypossessing a faster self renewal kinetics profile. Some reports indicatethat although the LTR HSC is the “true” stem cell, this stem cell is notcapable of lethal radiation rescue when transplanted to secondarylethally irradiated hosts without the presence of (10,65). Thephenomenon is generally attributed to the slow kinetics of self renewalof the “true” LTR HSC subset. However, it is possible that theIL-12-responsive subset possesses a faster kinetic self-renewal profile,leading to lethal radiation rescue in the absence of faster-acting,short-term repopulating (STR) HSC.

H. Function of “Survival” Stem Cells

The data indicate that the IL-12-responsive, survival-related, stemcells functions as long-term repopulating hematopoietic stem cells. Thedata further suggest that IL-12-responsive LTR HSC are expanded by thedirect action of IL-12. An alternative explanation is that theIL-12-responsive HSC are actually short-term repopulating cells thatconfer survival, or support, to long-term repopulating cells. Anotherpossible explanation would be that the IL-12 responsive cells are nothematopoietic stem cells, but are another stem cell type that conferssurvival to long-term repopulating HSC. Indeed, given any of thesescenarios, the IL-12-responsive subpopulation is an important subset ofsurvival-related stem cells that are invaluable in numerous clinicalscenarios, including ex vivo expansion of HSC LTR.

The isolation of a primitive, quiescent, IL-12-responsive HSC isimportant in hematopoietic stem cell biology, having implications forhematopoietic diseases and cancer therapy. As mentioned above, as asurvival-conferring stem cell subpopulation, the IL-12-responsivesubpopulation may be responsible for repopulation of HSC under stresses,such as radiation injury, chemotherapy or surgery.

It has been reported that stem cells can be lured to the site of tumorformation via cytokines secreted by breast cancer, wherein the stemcells are utilized by the tumor to promote tumor growth (67). Anintriguing scenario is that the IL-12-responsive, survival-conferringstem cells can also home to sites of tumor progression followingactivation by IL-12, but instead of promoting tumor growth, the IL-12responsive stem cells promote tumor destruction. This hypothesis isexpected to be borne out given the known anti-tumorigenic andanti-angiogenic properties of IL-12. In support of this notion, IL-12 isobserved to be down regulated by cancer cells, which fosters animmunosuppressive environment for tumor proliferation (68,69). Furthersupport for this notion is found in a report indicating that IL-12 canmobilize hematopoietic cells (70).

Finally, because the IL-12 responsive HSC subpopulation conferssurvival, and can be expanded in vivo with or without the stress ofradiation injury, it is a very attractive HSC subpopulation for ex vivoHSC expansion. Thus, the use of the novel, IL-12-responsive HSCsubpopulation can fill the clinical void for expanded HSC and theirrespective differentiated blood products.

I. The IL-12/IL-12R System as a Master Regulator of Hematopoiesis:

The data presented herein show that the IL-12/IL-12R ligand and receptorsystem represents a master regulator of hematopoiesis involved in theregulation of survival-related mechanisms. First, on the functionallevel of the organism, the administration of exogenous IL-12 is shown torescue mice from a lethal onslaught of radiation (FIG. 1). The fact thatIL-12 can rescue mice from lethal irradiation with near equaleffectiveness when administered either before or after radiation shows adirect effect of IL-12 on cells within the bone marrow (FIG. 1). Inaddition, the IL-12 facilitated lethal radiation rescue and results incomplete reconstitution of all blood groups, i.e., white blood cells,red blood cells and platelets (FIG. 2). These data show that the IL-12ligand acts at an IL-12 receptor on HSC. Second, the data also show thatexogenous IL-12, administered at the time of lethal radiation, rescueslong-term repopulating (LTR) HSC (FIG. 4). The data also show thatadministration of exogenous IL-12 increases the number of cells that donot express lineage markers (lineage negative cells; Lin⁻) but doexpress the IL-12 receptor (Lin³¹ IL-12R⁺ cells) as a cell surfacemarker. Lineage negative cells isolated from bone marrow largelycomprise immature (undifferentiated) cells, containing a mixture ofshort-term repopulating (STR), long-term repopulating (LTR) andprogenitor cells. Lineage negative cells are about 20-fold enriched inHSC as compared to whole bone marrow. Thus, the data show that amonglineage negative cells, cells bearing the IL-12 receptor are “expanded”following treatment with exogenous IL-12. The competitive repopulationstudy using isolated and selected IL-12R⁺ and IL-12R⁻ lineage negativecells further suggest that the “expansion” of Lin⁻ IL-12R⁺ cellsrepresent expansion of LTR HSC (Table 4). Thus, taken together, the datasuggest that the IL-12/IL-12R system is directly integral to processesof hematopoiesis and is a decisive factor in HSC preservation.

The IL-12 ligand is mainly produced in dendritic cells (43,44) thatcirculate in blood and are also resident in various tissues throughoutthe body (88). Generally circulating dendritic cells are referred to asmonocytes and tissue-specific dendritic cells are referred to asmacrophages. These dendritic cells are known as the “sentinel” cells ofthe body capable of sensing “stresses or threats” that relate to thesurvival needs of the organism (88). These “stresses or threats” come inthe form of infections by virus and bacteria and injury and wounds,including loss of blood cells, e.g. blood cell loss following radiationdamage or surgery. For example, when a lipopolysaccharide (LPS) moleculefrom an invading organism binds to a particular toll-like receptorslocated on the surface of dendritic cells, the IL-12 ligand is a directproduct of this activation. It is in this manner that dendritic cellscan “sense” “stresses or threats” to the organism (89). One of thecritical responses of these “sentinel” cells is to increase theproduction of the IL-12 ligand to combat the “stress or threat” (88,89).Thus the IL-12 ligand can be viewed as a “danger signal” indicating thesensing by and subsequent activation of the “sentry” dendritic cells.Moreover, the IL-12 receptor on stem cells, which is activated by theIL-12 danger signal, provides a mechanism for alleviating the danger orthreat to survival of the organism.

In the context presented herein, an illustrative example is thesignaling of “danger” in an organism following a non-lethal dose ofradiation or wounding (96). Cellular debris, such as native DNAresulting from cell lysis of blood cells, can serve to activatedendritic cells resulting in production of the IL-12. ThisIL-12-mediated danger signal is then communicated to cells within thebone marrow compartment as a warning sign to produce more blood cells.Further, the data show that this danger signal in the form of the IL-12ligand is directly communicated to HSC bearing the IL-12 receptor“instructing” these cells to expand in the face of impending danger (seeExample 13 below). HSC identified by the presence of the IL-12 receptorare a subset of survival-responsive HSC that can rapidly be induced toexpand under stress. An illustration of the master regulator function ofIL-12 is depicted schematically in FIG. 5.

Following lethal radiation, these sentinel cells are affected by theradiation and can no longer produce sufficient quantities of IL-12 torescue MSC residing in the bone marrow compartment. Thus, the exogenousaddition of the IL-12 is necessary as a replacement for the impairedendogenous production of the IL-12 danger signal. With exogenousreplacement of IL-12, HSC bearing the IL-12 receptor are induced toexpand, thereby conferring survival to the organism in the face oflethal radiation.

The IL-12/IL-12R system represents a novel approach to the elucidationof HSC biology and preservation. This system not only allows elucidationof mechanisms related to HSC expansion, but also provides a functionalsystem at the level of the organism to gauge HSC properties. Finally, asit relates to HSC, the IL-12/IL-12R system can provide a rapid inroad toa more focused understanding of stem cell biology, thereby advancing themanipulation of stem cells for clinical advantages.

J. Tissue Expression of IL-12Rβ2

The IL-12Rβ2 protein is expressed in a variety of tissues in the humanbody outside of the hematopoietic system, These cell populationsrepresent sources of tissue-specific progenitor cells that could alsoproliferate and reconstitute the cell population in the presence ofIL-12. Exogenous IL-12 could also provide repair of damaged or diseasedtissue. One example is damage caused by chemotherapy or radiation.

Immunohistochemistry for IL-12Rβ2 shows that the receptor subunit isstrongly expressed in the brain, specifically neuronal cells of thecerebral cortex, hippocampus, lateral ventricle, and the cerebellum. Seethe Human Protein Atlas entry on the world wide web for “IL12RB2”. TheHuman Protein Atlas is described in Nat Biotechnol., 28(12):1248-50(2010). Hepatocytes of the liver show weak staining for IL-12Rβ2, whileglandular cells of the gall bladder exhibit moderate staining intensity.In the gastrointestinal tract, the stomach, duodenum, small intestine,appendix, and rectum show moderate staining for IL-12Rβ2. Othergastrointestinal regions show weak staining, such as the oral mucosa,salivary gland, esophagus, lymphoid tissue of the appendix, and thecolon. Weak staining for IL-12Rβ2 has been shown in the nasopharynx,bronchus, and macrophages of the lung. Myocytes of the heart muscle alsoshow weak staining. In the female reproductive system, the uterus showsmoderate expression of IL-12Rβ2, while weak expression is exhibited inthe breast, uterus, and fallopian tube. Decidual cells of the placentashow moderate expression of IL-12Rβ2. while trophoblastic cells of theplacenta show weak expression. In the male reproductive system, moderateexpression of IL-12Rβ2 is seen in leydig cells of the testis, while weakexpression is seen in the epididymis, prostate, and seminal vesicle.Tubules in the kidney show moderate staining, while weak staining forIL-12Rβ2 is seen in the bladder. Skin also shows weak staining forIL-12Rβ2. Moderate IL-12Rβ2 staining is seen in the parathyroid andadrenal glands, while the thyroid exhibits weak staining for IL-12Rβ2.

Gastrointestinal tract: IL-12Rβ2 expression is detected on both panethcells and gastrointestinal stem cells on the crypt base of the smallintestine. This observation is significant because the intestinal cryptpaneth cells are responsible for maintenance of host defense in theintestine. Intestinal stem cells are responsible for the long-termmaintenance of the intestine by constant replenishment of epithelial andgoblet cells that makeup the intestinal villi. The close association ofpaneth cells with intestinal stem cells is thought to confer protectionof a critical stem cell population from hostile pathogens. FIGS. 6 a and6 b illustrate IL-12Rβ2 expression in the intestinal crypts of normaljejunum in humans and rhesus monkeys respectively. IL-12Rβ2⁺ intestinalcrypts are indicated by arrows. Intestinal crypts are circled.Intestinal stem cells are interspersed between crypt cells anddemonstrate characteristic “wedge shaped” morphology describedintestinal crypt cells.

Salivary glands: IL-12Rβ2 is expressed in normal human salivary glands.FIGS. 7A and 7B illustrate IL-12Rβ2 staining on normal serous secretoryunits (acini; labeled “AC”) and excretory ducts (labeled “ED”) of theparotid salivary gland in humans. The salivary acini secrete saliva anddigestive enzyme into the oral cavity via excretory ducts. Theimplication is that hyposalivation (xerostomia) is a frequent anddebilitating side effect or exposure to radiation and chemotherapy ofhead and neck cancer. There is a role for IL-12 in protection of seroussecretory units from radiation-induced xerostomia.

The invention is further described by reference to the followingexamples, which are provided for illustration only. The invention is notlimited to the examples, but rather includes all variations that areevident from the teachings provided herein. All publicly availabledocuments referenced herein, including but not limited to U.S. patents,are specifically incorporated by reference.

K. Therapeutic Administration of IL-12

IL-12 can be used therapeutically as a stand-alone drug (i.e. withoutprior, concurrent, or post-administration use of therapeutic cellpopulations) to confer regeneration and healing in multiple tissues ororgans.

The specific dose and administration of IL-12 is important fortherapeutic regenerative actions of IL-12. For example, hepaticregeneration can be adversely effected by continuous IL-12administration (IL-12 induces specific cytotoxicity against regeneratinghepatocytes in vivo; Matsushita et al., International Immunology11:657-665 (1999).

Regeneration of the bone marrow after radiation-induced myeloablationcan be achieved to the degree that the animal is rescued from the tissuedamage and survives, as shown in examples 1 and 17 in mice, and Example18 in rhesus monkeys. Example 15 illustrates how IL-12 administrationalone after myeloablative radiation can regenerate bone marrow andreconstitute the hematopoietic system as well as bone marrow transplant,i.e. the therapeutic use of cells for regeneration.

IL-12 receptors are expressed in many cell types in addition tohematopoietic and immune system cells of the bone marrow and circulatingblood such as brain tissue, lung tissue, kidney tissue, pancreatictissue, liver tissue, or cardiac tissue and the like. The cell typesinclude stem cells, progenitors and other cells of more mature andmature lineages.

IL-12 receptors are also expressed on non-stem cells that areco-localized with stem cells in given tissues. These cells act assupport for regeneration and contribute to the regenerative process.IL-12 modulation of these cells acts to further support the totality ofthe regenerative process.

IL-12 acts as a central actor in the regenerative system of mammals inresponse to injury and concomitant infections that occur with injury.Hence it is a general regenerative factor in the body.

The actions of IL-12 are not limited to cells wherein receptorexpression can be clearly observed at basal levels, as is the case inmany tissues. Expression of the IL-12 receptor is induced by injury, andthus after injury, cells expressing the IL-12 receptor become amenableto modulation by IL-12 and can participate in the act of the healing andregeneration.

Stem cell markers are co-expressed with the IL-12 receptor on asubpopulation of cell types. These markers (e.g. CDCP1, c-kit, KDR,Flt3, SLAM, CD133, IFNGR) are stem cell markers in numerous cell types(e.g. CD133 as a neural stem cell marker) and cells in body tissuesexpressing these markers are involved in regeneration of damaged tissue.

IL-12 administration is not limited to acting on only currently definedstem cell marker marked cells but on cells with still unknown stem cellmarkers.

IL-12 receptors are also expressed on non-stem cells that areco-localized with stem cells in given tissues. These cells act assupport for regeneration and contribute to the regenerative process.IL-12 modulation of these cells acts to further support the totality ofthe regenerative process.

The specific dose and administration of IL-12 is important fortherapeutic regenerative actions of IL-12. For example, hepaticregeneration can be adversely effected by continuous IL-12administration (IL-12 induces specific cytotoxicity against regeneratinghepatocytes in vivo; Matsushita et al International Immunology11:657-665 (1999).

IL-12 can aid in healing after stroke due to ischemia, thrombosis,hemorrhage or other cause. IL-12 can be administered alone to regeneratedamaged neural tissue after stroke, as well as after traumatic braininjury or following spinal cord injury. IL-12 can be administered to thenervous system using a variety of methods, including, for example:parenteral systemic administration; subcutaneous, intravenous,intraarterial, intramuscular or intraperitoneal administration; directcerebrospinal administration (epidural or peridural); intracerebral orintracerebroventricular administration. Spinal cord injuries can betreated with IL-12 by systemic administration as described above andalso by intrathecal administration (spinal canal administration).

Hematopoietic disorders of the bone marrow such as idiopathicthrombocytopenic purpura (ITP) or myelodysplastic syndrome (MDS) can betreated with administration of IL-12.

Therapeutic use of IL-12 can address hepatic damage due to injury ordisease state; pancreatic damage due to injury or disease state; heartdamage due to injury or disease state; kidney damage due to injury ordisease state; GI damage due to injury or disease, immune, hematopoieticand circulatory damage due to injury or disease state and other organ ortissue damages due to injury or disease state.

Use of IL-12 to treat damage due to injury or disease state can beapplied to organs or tissues of the body including, for example, thecirculatory system, digestive system, endocrine system, excretorysystem, integumentary system, lymphatic system, muscular system, nervoussystem, reproductive system, respiratory system, skeletal system.

Depending on appropriate treatment modality, administration can belocal, systemic, parenteral or topical. Depending on need, route ofadministration may be enteric, epidural, intracerebral,intracerebroventricular, epicutaneous, intradermal, subcutaneous, nasal,intravenous,intraarterial, intramuscular, intracardiac, intraosseousinfusion, intrathecal,intraperitoneal, (infusion or injection into theperitoneum) e.g. peritoneal dialysis, intravesical, intravitreal,intracavernous, intravaginal, intrauterine, extra-amniotic, transdermal,transmucosal, rectal or other known route of administration known in theart.

EXAMPLES Overview:

The applicant conducted research that led to the discovery of theradiation survival effect of IL-12. These studies demonstrated thatendogenously secreted factors are sufficient for hematopoietic rescue ofa subject following lethal radiation. (71) The experimental scenario isas follows. Animals were first implanted with a TheraCyteimmunoisolation device (TID). Following implantation of the device, theanimals received a lethal-dose of radiation, and then normal hone marrowLin⁻ cells were loaded into the device (thereby preventing directinteraction between donor and recipient cells). For control animals, theimplanted device was loaded with PBS with or without other types ofcells. Animal survival was evaluated and stem cell activity was testedwith secondary bone marrow transplantation and flow cytometry analyses.

These experiments provided clues as to the nature of the endogenous,lethal radiation, survival factors. First, there was a temporal effectrelated to the time allowed for vascularization of the device followingsurgical implantation in relation to the radiation event. Thisobservation led to the hypothesis that secreted hematopoietic factorsinvolved in recovery from the local injury, produced at the site ofimplantation, might be responsible for the rescue effect. Sincepro-inflammatory and inflammatory factors are generally known to beelicited following injury (72-74), it was postulated that IL-12 might beone of the factors responsible for the protection and survival effect.This postulate was later borne out by the data presented herein.

The examples described below demonstrate that IL-12 administrationprovides a “survival effect” to lethally irradiated mice via the IL-12R⁺HSC. The IL-12-induced, hematopoietic “survival effect” via the IL-12R⁺HSC is specific in that it does not occur with other similar cytokines.The survival effect stems from the interaction of IL-12 with IL-12R⁺long-term repopulating hematopoietic stem cells, thereby providingsignificant clinical importance. IL-12 administration acting on theIL-12R⁺ yields USC hematopoietic recovery following lethal and sublethalradiation by attenuating the decrease in blood cell counts observedafter lethal and sublethal radiation. These examples also show that invivo pretreatment with the IL-12 ligand leads to an increase in therelative number of IL-12R⁺ cells isolated from bone marrow andcompetitive repopulation studies indicate that IL-12R⁺ cells compriseLTR HSC. Taken together, these examples show that IL-12 acting via theIL-12R⁺ HSC is an in vivo hematopoietic stem cell protection andexpansion factor that allows for both rapid and broad range recovery ofhematopoiesis following hematological insult and that the IL-12 receptoris a novel hematopoietic stem cell marker.

Example 1 Low Dose of IL-12 Acting Via the IL-12R⁺ Stem Cell can ProtectMice from Lethal Ionizing Radiation

IL-12 (100 ng/mouse) was administered to C57BL/6J mice intravenouslyeither 24 hours before or 1 hr after lethal dose irradiation. Controlmice were irradiated and given PBS buffer. For mice given IL-12 beforeradiation, the survival rate is about 92%. For mice given IL-12following radiation the survival rate is about 78%. No control animalssurvived. In this example, IL-12 is effecting survival via interactionwith the IL-12 R⁺ stem cell (HSC). Survival data are shown in FIG. 1.

Example 2 IL-12/IL-12R Stem Cell Radioprotection Requires a Time Windowof Administration

The radiation rescue effect was tested at different times ofadministration of the IL-12, as described in Example 1. As shown inTable 2 below, approximately 100% or 80% rescue was achieved when 100 ngof IL-12 was administrated 24 hours before or 1 hour after total bodyirradiation, respectively. IL-12 was generally ineffective at the othertimes of administration tested. The time window for administration ofIL-12 along with radiation, particularly for administration afterradiation, suggests that the lethal radiation survival can only beeffected at about 24 hour intervals. When mice are given IL-12 12 hoursbefore radiation, the survival rate goes down. This indicates that theIL-12R⁺ stem cells are undergoing expansion and are no longer quiescent,and therefore more expanding stem cells are exposed to radiation. Thelower survival rate also indicates HSC expansion because it takes about24 hours to complete.

TABLE 2 RADIATION RESCUE OF MICE BY IL-12 INJECTION IL-12 administrationtime (hours) Con- trol −48 −36 −24 −12 +1 +12 +24 +36 Total 10 10 10 1010 10 5 10 5 mice # Survived 0 0 0 10 2 8 0 2 0 Survival 0 0 0 100 20 800 20 0 rate (%)

Example 3 IL-12/IL-12R⁺ Stem Cell Rescue Effect is Specific

Several cytokines related to IL-12 were also tested before and afterTotal Body Irradiation, as listed in Table 2. INF-γ has previously beenshow to be ineffective (51). Like IL-12, both IL-18 and IL-23 are knownto induce INF-γ production. IL-23 is in the same cytokine family asIL-12, sharing one subunit, namely the p40 subunit, and also sharing thebeta 2 subunit of the IL-12 receptor. IL-2 is also similar to IL-12 inthat it is an immuno-modulator of T cell differentiation. GM-CSF isknown to stimulate the proliferation of neutrophils and macrophageprogenitors.

All the cytokines were given at a dose of 100 ng/mouse, 30 to 60 minutespost-radiation. As shown in Table 3 below, the rescue effect is observedonly for IL-12 and IL-12/GM-CSF treated mice. Since GM-CSF alone was notobserved to provide protection, the protective effects observed for theIL-12/GM-CSF group are reflective of the protective effects of IL-12alone. These results demonstrate that the IL-12 protective effectappears to be specific, as these effects are not produced by any of theIL-12-related cytokines tested herein

TABLE 3 RADIOPROTECTION EFFECTS OF DIFFERENT CYTOKINES INJECTED AFTERTBI GM-CSF + Control GM-CSF IL-2 IL-18 IL-23 p40 IL-12 IL-12 Total miceNumber 5 4 5 4 5 5 5 5 Survived 0 0 0 0 0 0 4 4 Survival (%) 0 0 0 0 0 080 80

Example 4 Administration of IL-12 Acting Via the IL-12R⁺ Stem CellInduces Hematopoietic Recovery Following Lethal or Sublethal Radiationof All Major Blood Groups

In both animal toxicity studies and in clinical studies, high doseand/or repeated administration of IL-12 results in inhibition ofhematopoiesis, i.e., a decrease in the blood cell counts in peripheralblood, a decrease in bone marrow cellularity, and a decrease in colonyforming (CFC) cells are observed. Blood cell counts were examined inboth lethally irradiated (1000 rad or about 10 Gray) and sublethallyirradiated (500 rad or about 5 Gray) animals, as shown in FIG. 2. Aftera lethal dose of irradiation, there was no difference between theIL-12-treated and the non-treated animals in terms of peripheral bloodcounts during the first 12 days post-radiation. However, after 12 days,a rapid recovery of blood cell counts was observed in IL-12-treatedanimals, while all the control animals died (FIG. 2). Examination ofbone marrow cellularity revealed that at 9 days post-radiation, therewere 3 times more cells in the bone marrow of IL-12-treated animals thanin control animals (9.5×10⁵ vs. 3.1×10⁵). Histological examination ofthe bone marrow of the IL-12 treated animals showed that these animalsdisplayed a higher cellularity and better preservation of the bonemarrow structure as compared to control animals, as shown in FIG. 3.After sublethal irradiation, the blood cell counts in animals treatedwith IL-12 (either before or after radiation) showed less of a cellcount drop and an earlier recovery.

Example 5 IL-12 Acting Via the IL-12R⁺ Stem Cell Rescues IL-12R⁺Long-Term Repopulating (LTR) Hematopoietic Stem Cells

Most radioprotection studies observe animals for 30 days to determinethe survival effect. However, radiation-induced genomic instability andbystander effects result in damage that may affect the hematopoieticsystem long after the acute stage of radiation-induced injury. Only onestudy has carried out a long-term observation (300 days post-radiation)to assess the radioprotective effect of a combination of several growthfactors on hematopoiesis over the long-term (54). The viability andlong-term survival of the long-term repopulating hematopoietic stemcells was examined. Six months after a lethal dose of TBI, mice that hadsurvived, due to the protective effects of IL-12 treatment, weresacrificed, their bone marrow cells harvested, and 1×10⁶ donor bonemarrow cells (identified by the congenic marker Ly5.2) were transplantedinto lethally irradiated (950 rad) recipients (identified by thecongenic marker Ly5.1). Mice receiving the transplanted bone marrowcells outlived controls by at least 6 months.

Hematopoietic stem cells are a group of heterogeneous cells whichcontain long-term repopulating (LTR) HSC with complete self-renewalability, short-term repopulating (STR) HSC with limited self-renewalability, and uncommitted progenitor cells without self-renewal ability(10). Each of these subsets was examined to determine which compartmentsof HSC are protected by, or responsive to, IL-12 after lethal radiationusing assays of transplanted bone marrow cells. Bone marrow wastransplanted to secondary irradiated mice to test LTR HSC (FIG. 4A,D-F). Day 12 colony-forming units-spleen (CFU-S₁₂) from the micereceiving the transplant were assayed to examine STR HSC (FIG. 4B), andcolony-forming cells (CFC) assays were used to test for progenitor cells(FIG. 4C). Immediately after radiation (0 day), IL-12-treated bonemarrow had already lost CFU-S₁₂ and CFC activities. The recovery of LTRHSC activity in IL-12-treated bone marrow appeared by day 7 postradiation (FIG. 4A). STR HSC activity (CFU-S₁2) appeared almost fullyrecovered by day 10 after radiation (FIG. 4B), and progenitor cellactivity (CFC colonies) appeared recovered by day 14 (FIG. 4C). Thistime sequence of dynamic recovery of activities from 7 days (LTR HSC) to10 days (STR HSC) to 14 days (progenitor cells) matches the time courseof the normal HSC differentiation sequence from LTR HSC to STR HSC toprogenitors and is also correlated with expansion of mononuclearcolonies of IL-12-treated bone marrow at day 12 (FIG. 4B). Thus the STRHSC (CFU-S₁₂) and progenitor cell (CFC colonies) activities observedwere all derived from the protected and/or expanded LTR HSC. These LTRHSC recovered from IL-12-treated bone marrow 6 months after radiationrescued lethally irradiated mice in the long term and repopulated thecomplete hematopoietic system (FIG. 4D), including myeloid cells(14.1±1.4%; FIG. 4E), and lymphoid cells (15.1±3.1%; FIG. 4F). Theresults indicate that LTR HSC, but not STR HSC or progenitor cells, areprotected by IL-12 from radiation. Taken together with the results inTables 3 and 4, these data show that IL-12 is acting via the IL-12R⁺ LTRHSC.

Example 6 IL-12 increases the number of Sca-1⁺ HSC in vivo

By screening two known stem cell markers, namely Sca-1 and the Kitreceptor, we found that Sca-1⁺ cells were increased in IL-12-treatedbone marrow as compared with untreated bone marrow, 19.2% vs. 9.7%(p<0.05), respectively, at 24 hours post radiation. At day 7 postradiation, at which time LTR HSC showed repopulation activity, there wasas statistically significant greater number of Sca-1⁺ cells, 1.2×10⁵ vs.7.8×10⁴ (p<0.01), in IL-12-treated bone marrow as compared withuntreated mice. After lethal radiation, almost all c-kit marker cellswere depleted from bone marrow cells. There was no difference in thenumber of c-kit⁺ cells with or without IL-12 treatment. Further supportcomes from an early report that demonstrates the direct effect of IL-12on the expansion of HSC, which are lineage negative, Sca⁺ cells (92).

Example 7 Isolation of IL-12R⁺ Stem Cells from Bone Marrow

The IL-12 ligand has a direct effect on HSC expansion via the IL-12receptor present of LTR HSC. In addition, HSC expansion can lead eitherto the production of two daughter LTR HSC (symmetrical cell division) orone LTR HSC and one STR HSC (asymmetrical cell division), depending onthe status of the IL-12 ligand in circulation, i.e., concentration ofIL-12 and/or other factors (see schematic in FIG. 5).

Lineage negative IL-12R⁺ and IL-12R⁻ cells were isolated from bonemarrow and selected via FACS, and transplanted these cells into congenicrecipients. The goal of this experiment is to observe the donor cellcontribution for at least a period of at least four months, which wouldbe indicative of long-term repopulation. Herein, we report therepopulation data collected at various time points.

In Vivo Pretreatment of Mice with Exogenous IL-12 Directly Increases theNumber of IL-12R⁺ Cells Isolated from Bone Marrow in the Absence ofRadiation

C57BL/6 mice (6-8 weeks old) were either treated with exogenous IL-12(100 ng/mouse) via tail vein injection (Treated group) or not treated(Untreated group). Both groups were then sacrificed and their bonemarrow cells were harvested. In one group of mice (Group A), bone marrowwas harvested 25 hours after treatment with IL-12, and for another groupof mice (Group B), bone marrow was harvested at 21 hours after treatmentwith IL-12. Following isolation of whole bone marrow cells, both groupsof cells were depleted of lineage positive cells. For lineage positivecell depletion, bone marrow cells were fractioned to yield lineagenegative cells (Lin⁻) using lineage positive cell markers, namely, CD3e,CD4, CD5, CD8b, CD8a, B220, CD11b, Gr1 and Ter (80). Lin⁻ cells werethen collected using a magnetic cell sorting system. Next, lineagenegative cells were further fractionated via fluorescence-activate cellsorting (FACS) using the HAM10B9 antibody, which reacts with the β2subunit (IL-12Rβ2) of the mouse IL-12 receptor complex. FACS selectionto yield Lin⁻ IL-12R⁺ and Lin⁻ IL-12R⁻ cell populations. FACS analysis,shown in Table 4 below, revealed a 3.5 to 5.4 fold increase in Lin⁻IL-12R cells isolated from mice following treatment with IL-12 ligand.

TABLE 4 INCREASE IN LIN⁻ IL-12R⁺ CELLS ISOLATED FROM MICE TREATED WITHIL-12 LIGAND Treated Untreated Fold-Increase in Group Group Lin⁻ IL-12R⁺cells Group A (25 2.7% 0.5% 5.4 fold (after hour harvest) 25 hours)Group B (21 2.1% 0.6% 3.5 fold (after hr. harvest) 21 hours)

These data indicate that pre-treatment with exogenous IL-12 yields anincrease in the relative number of Lin⁻, IL-12⁺ cells among lineagenegative cells of the bone marrow compartment, and results in an average4.5 fold enhancement of IL-12R⁺ cells for the two different pretreatmenttime points. These data are also consistent with our BrdU incorporationassay in the absence of radiation, which showed an increase in BrdUpositive cells in whole bone marrow (16.5%) in IL-12-treated mice ascompared with untreated mice (7.5%) 21 hours after treatment. These datashow that the observed increase in isolated and selected lint IL-12Wcells from bone marrow following in vivo pretreatment with the IL-12ligand results from direct HSC expansion via the IL-12 ligand/IL-12receptor system, leading to an increase in the number of daughter HSCbearing the IL-12 receptor. This is consistent with the literature(90-93) and the data below.

Competitive Repopulation Using Lin⁻ IL-12R⁺ or Lin⁺IL-12R⁻ Isolated BoneMarrow Cells

For the competitive repopulation experiment, congenic donor andrecipient mice were used: donor mice (C57BL/6) bearing the Ly5.2 (CD45.2or ptprc^(b) allele) genetic marker and recipient mice (C57BL/6) bearingthe Ly5.1 (CD45.1 or ptprc^(a) allele) genetic marker. Cells fromtreated and untreated donor mice generated from Example 7 above (Lin⁻IL-12R⁻ and Lin⁻ IL-12R⁺) were each transplanted into lethallyirradiated recipients (950 rad) within 3 hours of radiation. Each groupof transplanted cells were supplemented with competitor cells in a 1:375ratio of selected cells to competitor cells.

Recipient Ly5.1 mice were separated into five groups: Group 1 received atransplant containing Lin⁻ IL-12R⁺ cells isolated from treated donorLy5.2 mice, as described above, Group 2 received a transplant containingLin⁻ IL-12R⁺ cells isolated from untreated Ly5.2 donor mice, also asdescribed above; Groups 3 and 4 received a transplant containing Lin⁻IL-12R⁻ cells from treated or untreated donor Ly5.2 mice respectively.Group 5 received Lin⁻ IL-12R⁻ cells from treated donor mice (the same asGroup 1), but also was subsequently treated with exogenous IL-12 (100ng/mouse via tail vein) 3 days after radiation and transplantation.Peripheral blood cells from mice in each group were sampled at five timepoints and subjected to FACS analysis for the marker Ly5.2 to determinethe percentage of donor cells. The data are shown in Table 5 below,which gives the % donor cell contribution, as measured by FACS analysisof peripheral blood cells for the Ly5.2 (CD45.2) marker at five timepoints.

TABLE 5 % DONOR CELLS IN PERIPHERAL BLOOD AT TIME PERIODS POSTTRANSPLANT % Donor Contribution 32 40 75 97 6 Days days days days*months Group 1 T IL-12R⁺ 2.5 2.5 2.2 1.4 5.0 Group 2 U IL-12R⁺ 8.8 3.01.5 0.8 9.2 Group 3 T IL-12R⁻ 16.8 1.7 2.0 0.9 3.6 Group 4 U IL-12R⁻48.3 2.2 2.6 1.0 10.0 Group 5 T IL12R⁺, +IL12 58.8 2.4 2.1 1.7 13.8*Animals were taken off antibiotics around day 92.

Treated groups receiving transplants of IL-12⁺ and IL-12⁻ cells (Groups1 and 3) show less peripheral blood repopulation by short termrepopulating cells (STR) than Untreated groups (Groups 2 and 4). In thisExample, STR includes differentiated cells. Comparison of UntreatedGroup 1 to Untreated Group 5 indicates that exogenous IL-12 followingthe transplant of Lin⁻ IL-12R⁺ cells leads to a rapid and significantincrease in STR; these comparative data further indicate the directexpansion of Lin⁻ IL-12R⁺ by the IL-12 ligand resulting in a largenumber of STR donor cells. A sharp drop in the STR cells counts wasobserved in donor peripheral blood cells around the 40 day mark for allgroups except Group 1; this sharp drop in peripheral blood cells hasbeen observed previously for LTR HSC and indicates the demise of STRcells (10). Group 2 had a large amount of change in the percentage ofdonor cells between 40 days and 6 months post-transplantation, whileGroup 1 has relatively little change over the same time period, showingthat Group 2 (untreated Lin⁻ IL-12R⁺ cells) had more STR cells thanGroup 1 (treated Lin⁻ IL-12R⁺ cells). Only treated Group 1 showed nochange in repopulation between sampling days 40 and 75, indicating thatpretreatment with IL-12, followed by selection of Lin⁻ IL-12R⁺ cellsyields predominately LTR HSC. Untreated Lin⁻ IL-12R⁺ cells (Group 2)show the smallest negative fold-change in donor cells from peripheralblood, indicating that Group 2 is less enriched in LTR HSC than Group 1,but significantly more enriched in LTR HSC than its counterpart Group 4,or even treated group 3. Overall, the significant differences betweentreated and untreated groups show that pretreatment with IL-12 leads toexpansion of LTR HSC in the bone marrow compartment, mobilization of STRinto the peripheral blood, and subsequent differentiation. Thus, Lin⁻cells isolated following pretreatment with IL-12 ligand arecomparatively enriched in LTR HSC.

Taken together, the data indicate that Lin⁻ IL-12R⁺ cells are present inthe bone marrow as both LTR HSC and STR HSC cells under “low” IL-12conditions (untreated groups). Upon pretreatment or post treatment withexogenous IL-12, a “high” status of the IL-12 danger signal isgenerated, whereby HSC are expanded and the STR HSC are mobilized awayfrom the bone marrow and into peripheral blood as previously reported inJackson et al. (70). This scenario is consistent with the presence ofhigh levels of the IL-12 danger signal as the organism is preparing tocombat the “stress or threat” associated with the danger signal. It isalso likely that under a “high” IL-12 status, mobilization of IL-12R⁺STR HSC also leads to differentiation. Referring to the schematic inFIG. 5, the data further indicate that following the “highly active” HSCstate of expansion, mobilization and differentiation resulting from a“high” IL-12 danger signal, if the status of the IL-12 danger signalbecomes “low,” the LTR HSC enter the quiescent state. The data in Table4 suggest that a IL-12 “low” status results following the subsequentisolation, purification and transplantation of IL-12R⁺ into lethallyirradiated recipients. Moreover, as discussed, even followingtransplantation, lethal radiation leaves the recipient in a state ofIL-12 “low” because the main source of IL-12, the sentinel dendriticcells, are initially lost until reconstitution via the transplant canoccur.

Although the dose of IL-12 used as the in vivo pretreatment or posttreatment is considered a “low” exogenous dose, this dose is probablyat, or higher than, what would be high levels of endogenously generatedIL-12 under various “stresses or threats” to the organism. It is alsonoteworthy that under the “stress or threat” of cancer, IL-12 isgenerally down-regulated. This down regulation of IL-12 may be directlyrelated to the proliferation of cancer cells under what would beperceived by the organism as a lack of a danger signal and itssubsequent responses (94).

About 92 days post transplantation the mice were taken off antibiotics.Antibiotics are generally known to suppress hematopoiesis. FACS analysisperformed on day 97 showed a drop in the peripheral blood countsresulting from the transplanted labeled cells (CD45.2).

The results at 6 months show that the Lin⁻, IL-12R⁺ cells are truelong-term repopulating stem cell. After 6 months, the IL-12R⁺ stem cellsstill persist and produce peripheral blood cells at a higher rate thanwhen the donor stem cell was first transplanted. The persistence overtime is the hallmark of a true long-term repopulating stem cell.Moreover, the expected percentage of donor cells in peripheral bloodwould be expected to be about 0.2-0.3%. However, the actual percentagefor the IL-12R⁺ treated mice from Group 1 is about 25 times higher thanthe expected percentage donor contribution based on the ratiotransplanted donor cells, and for IL-12 treated mice in Group 5, thedonor contribution is about 35 times expectations. For the IL-12R⁺untreated mice from Group 2, the actual donor contribution is greaterthan 50 times the expected donor contribution. Importantly, theseresults also show that by first treating mice with the IL-12 ligand andthen isolating Lin⁻, IL-12R⁺ cells, the resultant population of stemcells is mainly only LTR HSC. This result could have significantclinical benefit in enabling the selection of IL-12R⁺ stem cells withlong-term repopulating ability.

Example 8 Effect of IL-12 Administration on Mouse IL-12R⁺ Stem Cells inBone Marrow and GI Tract

Femoral bone marrow from irradiated mice treated with vehicle orrecombinant murine IL-12 (rMuIL-12) 24 hours or more after TBI (LD30/30)were stained for IL-12Rβ2 and evaluated for histological signs ofrecovery from radiation-induced injury at 12 days post TBI. As acontrol, bone marrow from non-irradiated, untreated mice wascharacterized with the presence of IL-12Rβ2-expressing hematopoieticstem cells, identified by co-staining for Sca-1 (a murine stem cellmarker), immature megakaryocytes with lobulated nuclei surrounded by anarrow rim of cytoplasm, matured megakaryocytes with lobulated nucleiand voluminous cytoplasm, and myeloid progenitor cells in themetamyelocyte stage (FIG. 8 a).

Bone marrow from mice treated only with vehicle and subjected to anLD30/30 of TBI (8.0 Gy) was characterized with minimal signs ofhematopoietic regeneration and the complete lack of IL-12Rβ2-expressingcells after 12 days following irradiation (FIG. 8 b). In contrast, micetreated with various dosing regimens of rMuIL-12 showed varying levelsof hematopoietic reconstitution, which was characterized with thepresence of IL-12Rβ2-expressing myeloid progenitors, megakaryocytes, andosteoblasts (FIG. 8 c-f). Mice treated with rMuIL-12, which has beendemonstrated to not cross react with IL-12 receptor, showed some signsof regeneration, however, lacked megakaryocytes (FIG. 8 g). For micetreated with rMuIL-12, however, no increase in the survival wasobserved, as compared with the vehicle control group.

In order to further evaluate as to whether morphologically identifiedcells were indeed hematopoietic stem cells and osteoblasts, bone marrowtissue sections were stained for the corresponding markers Sca-1 (forhematopoietic stem cells) and osteocalcin (for osteoblasts). As depictedin FIG. 9, IL-12Rβ2 expression was observed on cells that weremorphologically identified as hematopoietic stem cells and osteoblasts,which expressed Sca-1 (FIG. 9A) and osteocalcin (FIG. 9B), respectively.

Similar to hematopoietic stem cells and osteoblasts in femoral bonemarrow, mice jejunal crypts of the gastrointestinal tract expressedIL-12Rβ2 (FIG. 10 a). In the absence of irradiation, rMuIL-12administration at doses up to 200 ng/mouse did not cause injury injejunal crypts (FIG. 10 b, upper panels). Exposure to TBI (8.6 Gy),however, resulted in substantial jejunal damage 3 days afterirradiation, as evidenced by the widespread expression of LGR5, a GIstem cell marker that is expressed upon GI injury. Remarkably,administration of rMuIL-12 at the low dose range of 10 ng/mouse to 40ng/mouse dose-dependently mitigated radiation-induced jejunal damage,with no LGR5 expression evident at the optimal, efficacious dose of 20ng/mouse (FIG. 10 b, lower panels). In contrast, rMuIL-12 at the highdose of 200 ng/mouse exacerbated jejunal injury (FIG. 10 b, lower panel;tissue is counterstained to show structure; arrows point to LGR5positive cells). As observed with the rMuIL-12 dose ranges for optimalincreases in survival, these data show a window of opportunity formitigation of radiation injury by rMuIL-12 in a very low dose range ofthe drug that is also effective in alleviating bone marrow damage.

Example 9 Expression of IL-12R⁺ Stem Cells in Primate Bone Marrow and GITract

The expression of IL-12Rβ2 in non-irradiated rhesus monkeys and humanfemoral bone marrow and jejunum/ileum was evaluated byimmunohistochemistry. As depicted in FIG. 11A, non-human primate, aswell as human, progenitor cells and megakaryocytes expressed IL-12Rβ2.The expression of IL-12Rβ2 was also found on osteoblasts/osteoclastsfrom the bone marrow.

In the small intestine, IL-12Rβ2 was most commonly expressed in crypts(FIG. 11B). It is not known if IL-12Rβ2-expression in the intestinalcrypt is localized to Paneth cells, multipotent stem cells, or both.IL-12Rβ2 expression was also noted in lymphoid cells populating thelamina propria and submucosal regions (FIG. 11B). Mucin secreting gobletcells did not express IL-12Rβ2. Both crypt and lamina propriaIL-12Rβ2-expressing cells represent multifunctional mesenchymal-originmyofibroblasts that can serve as crypt shape-forming cells that alsooccupy both a stem cell niche and act as non-professional antigenpresenting cells to immunomodulatory cells in the lamina propria.

Example 10 Effect of IL-12 Administration on Non-Human Primate SurvivalFollowing Irradiation

The percentage of survival of rhesus monkeys exposed to an LD50/30 oftotal body irradiation (TBI) (6.7 Gy; 40 monkeys studied) was determinedfollowing subcutaneous administration of 100 ng/Kg or 250 ng/Kg ofrecombinant human IL-12 (rHuIL-12). The dosages of rHuIL-12 wereadministered at 24 hours post-TBI as well as both 24 hours and 7 dayspost-TBI. The study was conducted in the absence of any supportive care,including antibiotics. The doses of rHuIL-12 were chosen based on PK/PDstudies in rhesus monkeys and were equivalent to rMuIL-12 doses of 8ng/mouse and 20 ng/mouse, respectively. Animals were monitored forsurvival up to 30 days. One animal was excluded from the study due to abroken tooth.

As is depicted in FIG. 12A, rHuIL-12 at both doses, following eithersingle or two administrations, mitigated death due to irradiation to thesame extent. In each of the rHuIL-12-treated groups, survival increasedby 21% for mice receiving 100 ng/kg rHuIL-12 at 24 hours, and 25% overcontrol for all other treatement groups. Overall percentages of survivalwere 71% in the 100 ng/Kg single dose group (n=7) and 75% in all othergroups receiving rHuIL-12 (n=8) compared to 50% in the vehicle group.Between-group differences in percentage of survival were notstatistically significant, most likely because of the small number ofanimals in each group (n=8), but also because both rHuIL-12 doses werelikely within the efficacious dose range. However, analysis of thepercentage of survival regardless of the rHuIL-12 dosing regimenindicated that when pooled together, monkeys treated with rHuIL-12 hadsignificantly higher percentage of survival than those receiving vehicle(75% vs. 50%, respectively; P=0.05) (FIG. 12B).

Example 11 Effect of IL-12 Administration on Blood Cell Counts ofNon-Human Primates Following Irradiation

Rhesus monkeys were treated as described in Example 10. Blood sampleswere withdrawn from the monkeys at various times, and leukocytes andplatelets (thrombocytes), were counted by an automated hematologyanalyzer. Three analyses were conducted to assess differences in bloodcell counts between the treated and control groups during the studyperiod.

In one analysis, blood cell counts were analyzed from day 1 up to day 30following irradiation. Monkeys treated with rHuIL-12 had significantlyhigher numbers of leukocytes and platelets at days 12 and 14, around theday of maximum decrease in blood cell counts (“nadir”), for the 100ng/Kg and 250 ng/Kg doses, as compared to animals treated with vehicle(FIGS. 13A and 13B).

In a second analysis, blood cell counts were analyzed from day 1 up today 14, the day before any animals died. Monkeys treated with rHuIL-12had higher platelets counts compared to animals treated with vehicle(P=0.079 for the 250 ng/Kg group and P=0.02 for the 100 ng/Kg twicedosing group) during nadir (days 12 to 14). Additionally, in comparisonto the vehicle group, animals treated with rHuIL-12 had significantlyhigher counts of leukocytes (P<0.01 for the 250 ng/Kg group and P<0.04for the 100 ng/Kg twice dosing group) and reticulocytes (P<0.04 for the250 ng/Kg group and P<0.001 for the 100 ng/Kg group) during nadir (days12 to 14). The same trend was apparent for neutrophil, basophil, andlymphocyte counts, but those counts did not reach acceptable levels ofstatistical significance.

In a third analysis, the number of animals that reached dangerously lowplatelet counts during the study was assessed. This analysis revealed adifference between the vehicle and rHuIL-12-treated groups in the numberof platelet counts dropping below a threshold level of 20,000platelets/4, a level generally necessitating platelet transfusion. Inthe rHuIL-12 250 ng/Kg group, only 4 out of 16 (25%) platelet counts atthe nadir (day 12 to day 14) dropped below the transfusion threshold ofless than 20,000 platelets/4 whereas 12 out of 15 (80%) platelet countsfor the vehicle animals were below the threshold level during the sameperiod of time (P=0.007).

Taken altogether, these analyses show that rHuIL-12 increasesleukocytes, platelet, and reticulocyte counts just prior to the days onwhich animals begin to die from radiation toxicity (day 13, FIG. 13A).Interestingly, vehicle-treated animals that survived up to day 30 alsohad quick recovery of blood cell counts, which were statisticallyindistinguishable from those in the rHuIL-12 groups. These findingsdemonstrate that mortality occurs in animals which do not show a strongblood cell recovery around the nadir days. The finding was confirmed bycomparing blood cell counts of animals stratified by the mortalitystatus, i.e. those surviving up to day 30 versus animals dying after day12. In this analysis, the blood cell counts on the day before death wastaken for animals that died after day 12. The comparison day for thesurviving animals in each group was the average day on which thedecedents in a particular group died (days 14 to 18). This analysisdemonstrated that, regardless of the particular treatment group, animalssurviving up to day 30 had significantly higher counts of platelets,neutrophils, leukocytes, reticulocytes, and lymphocytes than those thatdied after day 12 (P<0.001 to P<0.05). When compared by treatment group,animals treated with 100 ng/Kg rHuIL-12 had significantly higher countsof neutrophils, leukocytes, and lymphocytes than did those treated withvehicle in both survivors and decedent groups (P<0.001 for all threecell types). In addition, animals treated with 100 ng/Kg rHuIL-12 had anumerically higher platelet and reticulocyte counts. These findingsdemonstrate that rHuIL-12-induced increases in blood cell counts aroundnadir play a role in promoting survival following radiation exposure.

Example 12 Marker Expression on IL-12Rβ2⁺ Human Hematopoietic Stem CellsMethods

Isolation of Lineage Marker-Depleted Cells: Human bone marrow wasobtained from Lonza (Walkersville, Md.). Cells were diluted with MACSbuffer (Miltenyi Biotec; Auburn, Calif.) and filtered through a 70 μmcell strainer (VWR; San Francisco, Calif.) to remove cell clumps. 35 mlof the cell suspension was layered over 15 ml of Ficoll-Paque (GELifesciences; Piscataway, N.J.) and centrifuged at 445 g for 35 minutesat 4° C. to separate bone marrow mononuclear cells. The mononuclearfraction was collected, diluted with MACS buffer and centrifuged at 200g for 10 minutes to remove platelets. The cell pellet was resuspended inMACS buffer and incubated with monoclonal antibodies specific forlineage markers (Miltenyi Biotec; Auburn, Calif.). Lineage markerpositive cells were removed by magnetic bead depletion. Lineage depletedcells (Lin⁻) were analyzed by flow cytometry. CD34⁺ cells, purified fromhuman bone marrow were purchased from Lonza (Walkersville, Md.).

Flow Cytometric analysis: Human bone marrow Lineage negative (Lin⁻)cells purified by immunomagnetic selection were washed in DPBS(Invitrogen; Carlsbad, Calif.). Cells were incubated with labeledantibodies against human IL-12Rβ2-APC (allophycocyanin label; R&Dsystems; Minneapolis, Minn.) and against CD34-PE (phycoerythrin label;BD Biosciences; San Jose, Calif.) for 30 minutes at room temperature.Next, cells were washed twice and suspended in DPBS and analyzed on aMoFlow flow cytometer (Beckman Coulter, Inc./Dako, Inc.). Theappropriate isotype controls were included for each experiment. CD34⁺cells were similarly labeled with antibodies against CD34-PE andIL-12Rβ2-APC. In select studies, cells were selected for IL-12Rβ2 andCD34 markers by cell sorting.

Immunocytochemical analysis of CD34⁺ cells: CD34⁺ cells obtained fromLonza were fixed on slides treated with 5 μg/ml fibronectin. Cultureswere fixed in cold methanol for 10 minutes at −20° C. and blocked withBackground Sniper (Biocare Medical, Concord, Calif.) for 15 minutes.Cells were labeled with a rabbit polyclonal antibody against IL-12Rβ2(Sigma) and incubated at room temperature for 2 hours, followed byincubation with ImmPRESS reagent anti-rabbit IgG Peroxidase (VectorLaboratories, Burlingame, Calif.). Slides were incubated with ImmPACTAEC Peroxidase Substrate (Vector Laboratories, Burlingame, Calif.) for30 minutes and counterstained in Hematoxylin. The negative controlincluded cells fixed and treated with the same reagents without theprimary rabbit polyclonal antibody. Photographs were taken using anOlympus camera in combination with analysis software.

Results

To determine if human hematopoietic stem cells (HSCs) expressedIL-12Rβ2, Lineage depleted (Lin⁻) cells were isolated from human bonemarrow and stained with antibodies to IL-12Rβ2 and CD34 to quantify theexpression of IL-12Rβ2 by flow cytometry. Approximately 1-2% of Lin⁻cells from human bone marrow expressed IL-12Rβ2 (FIG. 14A). These Lin⁻IL-12Rβ2⁺ comprised approximately 0.2% of the total bone marrow.Approximately 6-50% of Lin⁻CD34⁺ expressing cells were positive forIL-12Rβ2 (FIG. 14B). The range of IL-12Rβ2 co-expression of CD34⁺ cellswas observed to be donor dependent.

The flow cytometry results were supported by immunocytochemical stainingof CD34⁺ cells from human bone marrow for IL-12Rβ2 using a differentantibody to IL-12Rβ2 than was used for flow cytometry. As shown in FIG.14C, IL-12Rβ2 is expressed on CD34⁺ hematopoietic stem/progenitor cellsfrom human bone marrow.

To analyze the co-expression of IL-12Rβ2 with other hematopoietic stemcell markers, lineage depleted and CD34⁺ cells from human bone marrowwere labeled with antibodies to IL-12Rβ2 and the hematopoietic stem cellmarkers CD34, ckit, KDR, CD133, Flt-3, SLAM and CDCP1 and analyzed byflow cytometry. FIG. 15A shows the percentage of lineage depleted,IL-12Rβ2 expressing cells co-expressing above mentioned HSC markers.Approximately 46% of lineage depleted, IL-12Rβ2 expressing cellsco-expressed CD34. Similarly, c-kit, KDR, CD133, Flt3 and SLAM wereassociated with IL-12Rβ2 expressing cells. FIG. 15B shows the percentageof CD34⁺ IL-23Rβ2 expressing cells co-expressing HSC markers.Approximately 70% of CD34⁺ IL-12Rβ2 expressing cells co-expressed c-kitand up to 80% of the CD34⁺IL-12Rβ2⁺ population expressed CDCP1. Asmaller percentage of this population was associated with markers suchas KDR, CD133, Flt3 and SLAM. The frequency of subpopulations withinIL-12Rβ2 is listed in Table 6 below.

TABLE 6 Frequency of subpopulations within Lin⁻ IL-12Rβ2⁺ and CD34⁺IL-12Rβ2⁺ cells Subpopulation phenotype Lin⁻ IL-12Rβ2⁺ CD34⁺ IL-12Rβ2⁺CD34 46-55 94.84 ckit 40-55 68.12 KDR 34-36 18.81 CD133 6.2 2.89 Flt30.7 4.29 SLAM 23.3 3.03 CDCP1 6 77.85

Example 13 Effect of Exogenous IL-12 on CD34⁺ Hematopoietic Stem CellsMethods

Culture and stimulation of CD34⁺ cells with IL-12: CD34⁺ cells obtainedfrom Lonza (Walkersville, Md.) were thawed and washed in 0.1% BSA/DPBS.They were centrifuged at 300 g for 10 minutes and pellet was resuspendedin IMDM/10% FBS containing BD Golgi Plug, Brefeldin A (BD Biosciences).Cultures were stimulated with 10 pM IL-12 or media and incubated for 24hours at 37° C. in a humidified incubator. Cells were harvested,resuspended in Fixation/Permeabilization buffer (BD Biosciences) andincubated at 4° C. for 20 minutes. The cells were then washed in BDPerm/Wash buffer and resuspended in DPBS. Cells were labeled withantibodies to CD34 and IL-12Rβ2 and incubated for 30 minutes at roomtemperature, then washed and analyzed by flow cytometry. The resultswere compared to unstained and isotype controls.

Clonogenic progenitor assay: Human lineage depleted bone marrow cells,selected for IL-12Rβ2 and CD34 were plated at a concentration of 3200cells onto 35 mm cell culture dishes in methylcellulose mediumcontaining 1% methylcellulose, 30% fetal bovine serum, 1% BSA, 10-4 M2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml recombinant stem cellfactor (SCF), 10 ng/ml GM-CSF, 10 ng/ml and 3 μg/ml recombinanterythropoietin (Epo) (Stem Cell Technologies). Cultures weresupplemented with varying concentrations of IL-12 or media alone andincubated at 37° C., in 5% CO₂, with at least 95% humidity. CFU-GEMMcolonies were scored on Day 8 by morphological analysis using aninverted microscope.

Results

To determine if IL-12 acts directly on hematopoietic stem/progenitorcells, CD34⁺ cells from human bone marrow were cultured with IL-12 ormedia alone in the presence of Brefeldin A for 24 hours. Cells wereharvested, fixed and permeabilized. Next, cells were labeled withantibodies to IL-23Rβ2 to quantify intracellular IL-12Rβ2 expression byflow cytometry analysis. Approximately 6% of CD34⁺ cells from human bonemarrow expressed IL-12Rβ2. Following stimulation with IL-12, IL-12Rβ2expression increased 3 fold to 21% (representative analysis shown inFIG. 16).

To test the clonogenic potential of IL-12Rβ2⁺ CD34⁺ cells, lineagenegative cells isolated from human bone marrow were labeled withantibodies to IL-12Rβ2 and CD34. Cells were sorted to obtain twoIL-12Rβ2 expressing populations: 1) IL-12Rβ2⁺CD34⁺ and 2) IL-12Rβ2⁺CD34⁻(FIG. 17A). Each of these populations was plated in methylcellulosecultures with or without IL-12 stimulation and examined for colonyforming units of myeloid cells (CFU-GEMM). CFU-GEMM colonies werecounted on Day 8 of culture. The colony forming potential of culturesstimulated with IL-12 was compared to the unstimulated control.IL-12Rβ2⁺CD34⁻ cells failed to form any colonies. One CFU-GEMM colonyobserved in IL-12Rβ2⁺CD34⁻ cultures stimulated with 1 nM IL-12 could beattributable to contamination during sorting (FIG. 17B). Interestingly,an increase in CFU-GEMM colony formation was observed in culturesstimulated with 50 pM IL-12. Increasing the IL-12 concentration to 1 nMresulted in inhibition of CFU-GEMM colony formation. A clear correlationexists between IL-12 stimulation and colony growth. However, colonynumbers and types were variable and highly donor-dependent.

Human bone marrow lineage-depleted cells could be further subdividedinto two subsets based on their forward and side scatter as shown inFIGS. 18A and 18B. Both of these populations together give CFU-GEMMcolonies along with some more committed colony types. Interestingly,lineage depleted cells from one specific donor appeared to be deficientin one subset (FIG. 18B). Lin⁻IL-12Rβ2⁺CD34⁺ cells from this donor gaveonly BFU-E colonies along with some committed progenitors, but noCFU-GEMM colonies. This shows that both subsets of the lineage-depletedpopulation were essential for generation of CFU-GEMMs.

Example 14 Therapeutic Application of the IL-12R⁺ Stem Cell

The Examples above show that the IL-12R⁺ stem cell has survival relatedproperties in generating hematopoiesis following a lethal radiationassault. The property of transdifferentiation has been attributed to thehematopoietic stem cells. Thus, given the survival related properties ofthe IL-12 stem cell, this cell can be used for repair and regenerationof tissue of various types other than blood.

For autologous stem cell repair and regeneration, the IL-12R⁺ stem cellsare isolated as described in example 7. The IL-12 ligand is given to thepatient prior to bone marrow or peripheral blood harvest of cells.Generally bone marrow is preferred as the source of blood cells forharvest. Whether or not the IL-12 ligand is given to the patient, thenext step is to select cells using an appropriate antibody that arepositive for the IL-12 receptor. In a preferred embodiment, a lineagepositive depletion step is performed first which would yield lineagenegative cells to use as the blood cell population for selection of theIL-12 receptor. Further refinement of the IL-12R⁺ stem cell populationis made using various antibodies, such as CD34, CD133, Kit, HSMI, Flt3R,IFNGR, TpoR, CD38, Angiotensin(1-7)R, CCR, CXCR and the like. Secondarymarker choice may differ depending on the type of tissue in need ofrepair. Following isolation of the IL-12R⁺ stem cell, the cells can becryogenically frozen, but it is preferred that the cells be used withoutfreezing as soon as possible following harvest. Then these cells can betransplanted back to the patient.

For example, in the case of a hematopoietic stem cell transplant,following harvest and isolation of the a population of IL-12R⁺ cells,the patient undergoes myeloablation and then receives the IL-12R⁺ stemcells with or without other carrier cells, which are blood cells in thiscase. An effective dose of the IL-12 ligand may also be givenimmediately following transplantation. An effective dose of the IL-12ligand is generally 500 ng/kg or less. Also following the HSCtransplant, the patient may be given an effective dose of the IL-12ligand. The post-transplant administration of the IL-12 ligand could bedone one or more times post-transplantation. It is preferred that theIL-12 ligand not be given for longer than 1 week post-transplant. Duringlong-term recovery from the transplant, the patient may periodically begiven a dose of the IL-12 ligand as needed.

For autologous liver repair and regeneration, the above described methodis followed. Liver specific stem cell markers are used as secondarymarkers of the IL-12R⁺ stem cell. In the case of liver repair andregeneration, a population of IL-12R⁺ stem cells are injected into theliver, with or without radiation specifically to the liver prior totransplantation. Once in the liver, the IL-12R⁺ stem cells facilitaterepair and regeneration of the liver. Under these conditions, theIL-12R⁺ cells undergo transdifferentiation to generate hepatocytes. TheIL-12 ligand may or may not be given to the patient followingtransplantation. It is preferred however that the IL-12 ligand isadministered for some time period following transplantation. In asimilar manner, cardiac, kidney, pancreas, lung tissue, or the like canrepaired and regeneration via transplantation of the IL-12R⁺ stem cells,with or without administration of the IL-12 ligand.

For allogeneic repair and regeneration of tissue, a population ofIL-12R⁺ stem cells is derived from a healthy person who is not thepatient. Selection of a suitable donor is made in the manner generallyuse for finding closely matched donor and recipient pairs. In the caseof allogeneic use of the IL-12R⁺ stem cell, IL-12R⁺ stem cells that lackone or more major histocompatibility markers (MHC) on the cell surfaceare selected. Following selection of the IL-12R⁺ stem cell population,antibodies to various MHC are used to select for a cell population thatis IL-12R⁺ and MHC negative. This population is preferred for allogeneictransplantation for tissue repair and regeneration of bone marrow,liver, cardiac, kidney, pancreas, lung tissue or the like.

Example 15 Reconstitution of Hematopoietic System in Mice Using IL-12

Studies were conducted to compare IL-12 administration to the use ofHSCT (healthy bone marrow cells) in generating hematopoietic recovery inlethally irradiated mice. All mice received an acute dose of lethalradiation (8 Gy). One group of lethally irradiated mice received IL-12administered as a split-dose at both 24 hours before and 48 hours afterirradiation. Another group of lethally irradiated mice received a bonemarrow transplant of 1×10⁶ healthy bone marrow cells.

Data from this study show that IL-12 alone recapitulates thehematopoietic recovery equal to that of a bone marrow transplant in bothits kinetic and blood cell parameters. The survival rate of IL-12treated mice at 21 days (about 70%) is similar to that of mice receivinga bone marrow transplant (about 90%; FIG. 19A). IL-12 treated mice hadabove normal neutrophil counts at 21 days (FIG. 19B), red blood cellcounts near the normal range at 21 days (FIG. 19C), and plateletrecovery in the normal range and above that of bone marrow transplantrecipients (FIG. 19D).

FIG. 20A shows a photomicrograph of bone marrow from an irradiatedcontrol mouse at 12 days post-irradiation. No cells are seen in the bonemarrow, typical of “empty marrow” seen in acute radiation syndrome. Incontrast, bone marrow from an IL-12 treated mouse at 12 dayspost-irradiation (FIG. 20B) contains megakaryocytes and foci ofregeneration (progenitors cells).

Example 16 IL-12 Treatment in Mice at Three Different Radiation Doses

Mice (n=10) were exposed to 8.6, 8.8 and 9.0 Gy doses of lethalirradiation which corresponded to LD70, LD90 and LD100, respectively.Twenty four hours following radiation exposure, 20 ng/mouse of rMuIL-12was administered subcutaneously and the mice were monitored for 30 daysurvival. FIG. 21A shows the Kaplan-Meier survival plots for the vehiclecontrol and rMuIL-12 treated mice for each of the three radiation doses.

rMuIL-12 administration yielded a similar statistically significantincrease in percent survival at each radiation dose. For theexperimental data depicted in FIG. 21A, rMuIL-12 administration yieldedstatistically significant increases in percent survival at eachradiation dose (via Kaplan-Meier analysis, (p<0.05−0.001 for increasingradiation doses; via chi squared (Fisher Exact) analysis, p<0.05 for allradiation doses). The efficacy of rMuIL-12 does not decrease withincreasing radiation exposure within the range of hematopoieticsyndrome. Moreover, the radiomitigation effects of rMuIL-12 actuallyseem to increase with increasing levels of radiation exposure. Thisobservation suggests that rMuIL-12 can ameliorate the gastrointestinal(GI) damage that is coincident with hematopoietic injury at higherradiation exposure.

Example 17 IL-12 Treatment in Mice at Three Different Times FollowingIrradiation

rMuIL-12 was administered subcutaneously (20 ng/mouse) to three groupsof mice (n=10) at either 24, 48 or 72 hours after LD100 radiationexposure (9.0 Gy). The Kaplan-Meier survival plot is shown in FIG. 21B.The 24 and 48 hour administrations resulted in statistically significantincreases in percent survival (p=0.01 for 24 hours and p<0.03 for 48hours, chi square analysis (Fisher's Exact Test)) vehicle control(p>0.05, K-M analysis).

The present invention has been discussed in considerable detail withreference to certain preferred embodiments, although other embodimentsare possible. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references cited herein are incorporated by reference intheir entirety.

The above examples are given to illustrate the present invention. Itshould be understood, however, that the spirit and scope of theinvention is not to be limited to the specific conditions or detailsdescribed in these examples. All publicly available documents referencedherein, including but not limited to U.S. patents, are specificallyincorporated by reference.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodifications and variations of this invention provided they come withinthe scope of the appended claims and their equivalents.

REFERENCES

-   1. Ogawa M. Differentiation and proliferation of hematopoietic stem    cells. Blood. 1993; 81:2844-2853.-   2. Williams D A. Ex vivo expansion of hematopoietic stem and    progenitor cells-robbing Peter to pay Paul? [editorial]. Blood.    1993; 81:3169-3172.-   3. Moore M A. Expansion of myeloid stem cells in culture. Semin    Hematol 1995; 32:183-200.-   4. Spangrude G J, Heimfeld S, Weissman I L. Purification and    characterization of mouse hematopoietic stem cells [published    erratum appears in Science. 1989, 244:1030]. Science. 1988;    241:58-62.-   5. Jones R J, Wagner J E, Cenalo P, Zicha M S, Sharkis S J.    Separation of pluripotent haematopoietic stem cells from spleen    colony-forming cells [see comments]. Nature. 1990; 347:108-189.-   6. Berardi A C, Wang A, Levine J D, Lopez P, Scadden D T. Functional    isolation and characterization of human hematopoietic stem cells.    Science. 1995; 267:104-108.-   7. Kawashima I, Zanjani E D, Almaida P G, Flake A W, Zeng H,    Ogawa M. CD34⁺ human marrow cells that express low levels of Kit    protein are enriched for long-term marrow-engrafting cells. Blood.    1996; 87:4136-4142.-   8. Huang S, Terstappen L W. Lymphoid and myeloid differentiation of    single human CD34⁺, HLADR⁺, CD38⁻ hematopoietic stem cells. Blood.    1994; 83:1515-1526.-   9. Kondo M, Wagers A J, Manz M G, Prohaska S S, Scherer D C,    Beilhack G F, Shizuru J A, Weissman I L. Biology of hematopoietic    stem cells and progenitors: implications for clinical application.    Annu Rev Immunol. 2003; 21:759-806.-   10. Zhao Y, Lin Y, Zhan Y, Yang G, Louie J, Harrison D E, Anderson,    W F. Murine hematopoietic stem cell characterization and its    regulation in BM transplantation. Blood. 2000; 96:3016-3022.-   11. Alamo A L, Melnick S J. Clinical application of four and    five-color flow cytometry lymphocyte subset immunophenotyping.    Cytometry. 2000; 42:363-370.-   12. Zhong I F, Zhao Y, Sutton S. Su A, Zhan Y, Zhu L, Yan C,    Gallaher T, Johnston P B, Anderson W F, Cooke M P. Gene expression    profile of murine long-term reconstituting vs. short-term    reconstituting hematopoietic stem cells. Proc Natl Acad Sci USA.    2005; 102:2448-2453.-   13. Park I K, He Y, Lin F, Laerum O D, Tian Q, Bumgarner R, Klug C    A, Li K, Kuhr C, Doyle M J, Xie T, Schummer M, Sun Y, Goldsmith A,    Clarke M F, Weissman I L, Hood L, Li L. Differential gene expression    profiling of adult murine hematopoietic stem cells. Blood. 2002;    99:488-498.-   14. Terskikh A V, Easterday M C, Li L, Hood L, Komhlum H I,    Geschwind D H, Weissman I L. From hematopoiesis to neuropoiesis:    evidence of overlapping genetic programs. Proc Natl Acad Sci USA.    2001; 98:7934-7939-   15. Gluckman E, Rocha V, Bayer-Chammard A. et al. Outcome of cord    blood transplantation from related and unrelated donors. Eurocord    Transplant Group and the European Blood and Marrow Transplantation    Group. N Engl J Med. 1997; 337:373-381.-   16. Wagner J E, Rosenthal J, Sweetman R, et al. Successful    transplantation of HLA-matched and HLA-mismatched umbilical cord    blood from unrelated donors: analysis of engraftment and acute    graft-versus-host disease. Blood. 1996; 88:795-802.-   17. Rogers I, Sutherland Holt D, et al. Human UC-blood banking:    impact of blood volume, cell separation and cryopreservation on    leukocyte and CD34⁺ cell recovery. Cytotherapy. 2001; 3:269-276.-   18. Lobato da Silva C, Goncalves R, Crapnell K B, Cabral J M S,    Zanjani E D, and Almeida-Porada G. A human stromal-based serum-free    culture system supports the ex vivo expansion/maintenance of bone    marrow and cord blood hematopoietic stem/progenitor cells. Exp.    Hematol. 2005; 3:828-835.-   19. Devine S M, Lazarus H M, Emerson S G. Clinical application of    hematopoietic progenitor cell expansion: current status and future    prospects. Bone Marrow Transplant. 2003; 31:241-252.-   20. McNiece I, Jones R, Bearman S I, et al. Ex vivo expanded    peripheral blood progenitor cells provide rapid neutrophil recovery    after high dose chemotherapy in patients with breast cancer. Blood.    2000; 96:3001-3007.-   21. Paquette R L, Dergham ST, Karpf E, et al. Ex vivo expanded    unselected peripheral blood: progenitor cells reduce post    transplantation neutropenia, thrombocytopenia, and anemia in    patients with breast cancer. Blood. 2000; 96:2385-2390.-   22. Reiffers J, Cailliot C, Dazey B, Attal M, Caraux J, Boiron J M.    Abrogation of post-myeloablative chemotherapy neutropenia by ex vivo    expanded autologous CD34-positive cells. Lancet. 1999;    354:1092-1093.-   23. Lewis I D, Almeida-Porada G, Du J, et al. Umbilical cord blood    cells capable of engrafting in primary, secondary, and tertiary    xenogeneic hosts are preserved after ex vivo culture in a noncontact    system. Blood. 2001; 97:3441-3449.-   24. Barker J N. Davies S M. DeFor T. Ramsay N K, Weisdorf D J,    Wagner J E. Survival after transplantation of unrelated donor    umbilical cord blood is comparable to that of human leukocyte    antigen-matched unrelated donor bone marrow: results of a    matched-pair analysis. Blood. 2001; 97:2957-2961.-   25. Wagner J E, Barker J N, DeFor T E, et al. Transplantation of    unrelated donor umbilical cord blood in 102 patients with malignant    and nonmalignant diseases: influence of CD34 cell dose and HLA    disparity on treatment-related mortality and survival. Blood. 2002;    100:1611-1618.-   26. Lazzari L, Lucchi S, Rebulla P, et al. Long-term expansion and    maintenance of cord blood haematopoietic stem cells using    thrombopoietin, Flt3-ligand, interleukin (IL)-6 and IL-11 in a serum    free and stroma free culture system. Br J Haematol. 2001;    112:397-404.-   27. Shpall E J, Quinones R, Giller R, et al. Transplantation of ex    vivo expanded cord blood. Biol Blood Marrow Transplant. 2002;    8:368-370.-   28. Barker J N, Weisdorf D J, DeFor T E, Blazar B R, Miller J S,    Wagner J E. Rapid and complete donor chimerism in adult recipients    of unrelated donor umbilical cord blood transplantation after    reduced-intensity conditioning. Blood. 2003; 102:1915-1919.-   29. Gluckman E, Broxmeyer H A, Auerbach A D, et al. Hematopoietic    reconstitution in a patient with Fanconi's anemia by means of    umbilical cord blood from an HLA-identical sibling. N Engl J Med.    1989; 321:1174-1178.-   30. Madlambayan G J, Rogers I, Kirouac D C, Yamanaka N, Muzurier F,    Doedens M, Robert Casper R F, Dick J E, and Zandstra P W. Dynamic    changes in cellular and microenvironmental composition can be    controlled to elicit in vitro human hematopoietic stem cell    expansion. Exp. Hematol. 2005; 33:1229-1239.-   31. Fitz K M, Ryan L. Hewick M, Clark R M., Chan S C, Loudon S C,    Sherman R, Perussia F, Trinchieri B G. Identification and    purification of natural killer cell stimulatory factor (NKSF), a    cytokine with multiple biologic effects on human lymphocytes. J Exp    Med. 1989; 170:827-845.-   32. Lertmemongkolchai G, Cai G, Hunter C A, Bancroft G J. Bystander    activation of CD8+ T cells contributes to the rapid production of    IFN-gamma in response to bacterial pathogens. Journal of Immunology.    2001; 166:1097-1105.-   33. Cui J, Shin T, Kawano T, Sato H, Kondo E, Toura I, Kaneko Y,    Koseki H, Kanno M, Taniguchi M. Requirement for Valpha14 NKT cells    in IL-12-mediated rejection of tumors. Science. 1997; 278:1623-1626.-   34. Ohteki T, Fukao T, Suzue K, Maki C, Ito M, Nakamura M, Koyasu S.    Interleukin 12-dependent interferon gamma production by CD8alpha⁺    lymphoid dendritic cells. J Exp. Med. 1999; 189:1981-1986.-   35. Airoldi I, Gri G, Marshall J D, Corcione A, Facchetti P,    Guglielmino R, Trinchieri G, Pistoia V. Expression and function of    IL-12 and IL-18 receptors on human tonsillar B cells. Journal of    Immunol. 2000; 165:6880-6888.-   36. Hsieh C S, Macatonia S E, Tripp C S, Wolf S F, O'Garra A, Murphy    K M. Development of TH1 CD4⁺ T cells through IL-12 produced by    Listeria-induced macrophages. Science. 1993; 260:547-549.-   37. Manetti R, Parronchi P, Giudizi M G, Piccinni M P, Maggi E,    Trinchieri G, Romagnani S. Natural killer cell stimulatory factor    (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific    immune responses and inhibits the development of IL-4-producing Th    cells. J. Exp. Med. 1993; 177:1199-1204.-   38. Brunda M J, Luistro L, Warrier R R, Wright R B, Hubbard B R,    Murphy M, Wolf S F, Gately M K. Antitumor and antimetastatic    activity of interleukin 12 against murine tumors. J. Exp. Med. 1993;    178:1223-1230.-   39. Noguchi Y, Jungbluth A, Richards E C, Old L J. Effect of    interleukin 12 on tumor induction by 3-methylcholanthrene. Proc    Natl. Acad Sci USA. 1996; 93:11798-11801.-   40. Giordano P N, De Giovanni N C, Landuzzi L, Di Carlo E, Cavallo    F, Pupa S M., Rossi I, Colombo N I P, Ricci C, Astolfi A, Musiani P,    Fomi G, Lollini P-L. Combined Allogeneic Tumor Cell Vaccination and    Systemic Interleukin 12 Prevents Mammary Carcinogenesis in HER-2/neu    Transgenic Mice. J. Exp. Med. 2001; 194:1195-1206.-   41. Colombo M P, Trinchieri G. Interleukin-12 in anti-tumor immunity    and immunotherapy. Cytokine Growth Factor Rev. 2002; 13:155-168.-   42. Yao L, Pike S E, Setsuda J, Parekh J, Gupta G, Raffeld M, Jaffe    E S, Tosato G. Effective targeting of tumor vasculature by the    angiogenesis inhibitors vasostatin and interleukin-12. Blood. 2000;    96:1900-1905.-   43. Ma X, Chow J M, Gri G, Carra G, Gerosa F, Wolf S F, Dzialo R,    Trinchieri G. The interleukin 12 p40 gene promoter is primed by    interferon gamma in monocytic cells. J. Exp Med. 1996; 183:147-157.-   44. Gazzinelli R T, Wysocka M, Hieny S, Scharton-Kersten T, Cheever    A, Kuhn R, Muller W, Trinchieri G, Sher A. In the absence of    endogenous IL-10, mice acutely infected with Toxoplasma gondii    succumb to a lethal immune response dependent on CD4⁺ T cells and    accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha. J.    Immunol. 1996; 157:798-805.-   45. Marcel E, Harry B. Radiation-induced apoptosis. Cell Tissue Res.    2000; 301: 133-142.-   46. Dewey W C, Ling C C, Meyn R E. Radiation-induced apoptosis:    relevance to radiotherapy. Int. J Radiat Oncol Biolo Phys. 1995;    33:781-796.-   47. Dubray B, Breton C, Delic J, Klijanienko J, Maciorowski Z, Vielh    P, Fourquet A, Dumont J, Magdclenat H, Cosset J-M. In vitro    radiation-induced apoptosis and early response to low-dose    radiotherapy in non-Hodgkin's lymphomas. Radiother Oncol. 1998;    46:185-191.-   48. Portielje J E A, Lamers C H J, Kruit W H J, Sparreboom A,    Bolhuis R L H, Stoter G, Huber C, Gratama J W. Repeated    Administrations of Interleukin (IL)-12 Are Associated with    Persistently Elevated Plasma Levels of IL-10 and Declining IFN-γ    Tumor Necrosis Factor-α, IL-6, and IL-8 Responses. Clin. Cancer Res.    2003; 9: 76-83.-   49. Eng V M, Car B D, Schnyder B, Lorenz M, Lugli S, Aguet M,    Anderson T D, Ryffel B, Quesniaux V F. The stimulatory effects of    interleukin (IL)-12 on hematopoiesis are antagonized by    IL-12-induced interferon gamma in vivo. J Exp. Med. 1995;    181:1893-1898.-   50. Car B D, Eng V M, Schnyder B, LeHir M, Shakhov A N, Woerly G,    Huang S, Aguet M, Anderson T D, Ryffel B. Role of interferon-gamma    in interleukin 12-induced pathology in mice. Amer. J Path. 1995;    147:1693-1707.-   51. Neta R, Stiefel S M, Finkelman F, Hellmann S, Ali N. IL-12    protects bone marrow from and sensitizes intestinal tract to    ionizing radiation. J Immunol. 1994; 153:4230-4237.-   52. Zsebo K M, Smith K A, Hartley C A, Greenblatt M, Cooke K, Rich    W, McNeice I K. Radioprotection of mice by recombinant rat stem cell    factor. Proc Natl Acad Sci USA 1992; 89:9464-9468.-   53. Drouet M, Mourcin F, Grenier N, Leroux V, Denis J, Mayol J F,    Thullier P, Lataillade J J, Herodin F. Single administration of stem    cell factor, FLT-3 ligand, megakaryocyte growth and development    factor, and interleukin-3 in combination soon after irradiation    prevents nonhuman primates from myelosuppression: long-term    follow-up of hematopoiesis. Blood. 2004; 103:878.-   54. Herodin F, Bourin P. Mayol J F, Lataillade J J, Drouet M.    Short-term injection of antiapoptotic cytokine combinations soon    after lethal y-irradiation promotes survival. Blood. 2003;    101:2609-2616.-   55. Streeter P R, Dudley L Z. Fleming W H. Activation of the G-CSF    and Flt-3 receptors protects hematopoietic stem cells from lethal    irradiation. Exp. Hematol. 2003; 31:1119-1125.-   56. Kucia M. Ratajczak J, Ratajczak M Z. Are bone marrow stem cells    plastic or heterogenous—That is the question. Exp. Hematol. 2005;    33:613-623.-   57. Meijne E I, van der Winden-van Groenewegen R J, Ploemacher R E,    Vos O, David J A, Huiskamp R. The effects of x-irradiation on the    hematopoietic stem cell compartment in the mouse. Exp Hematol. 1991;    19:617-623.-   58. McCarthy K. Population size and radiosensitivity of marine    hematopoietic endogenous long-term repopulating cells. Blood.    1997:89:834-841.-   59. Down J, Boudewijn A, van Os R, Thames H, Ploemacher R.    Variations in radiation sensitivity and repair among different    hematopoietic stem cell subsets following fractionated irradiation.    Blood. 1995; 86:122-127.-   60. Inoue T, Hirabayashi Y, Mitsui H, Sasaki H, Cronkite E P, Bullis    J E Jr, Bond V P, Yoshida K. Survival of spleen colony forming units    (CFU-S) of irradiated hone marrow cells in mice: evidence for the    existence of a radioresistant subtraction. Exp Hematol. 1995;    23:1296-1300.-   61. van Bekkum D. Radiation sensitivity of the hematopoietic stem    cell. Rad Res. 1991; 128:S4 8.-   62. Wagemaker G. Heterogeneity of radiation sensitivity of    hematopoietic stem cell subsets. Stem Cells. 1.995:13    (suppl):257-260.-   63. Zuniga-Pflucker J C, Kruisbeek A. M. Intrathymic radioresistant    stem cell follow an IL-2/IL-2R Pathway during thymic regeneration    after sublethal irradiation. J. Immunol. 1990; 144:3736-3740.-   64. Ayukawa K, Tomooka S, Asano T, Taniguchi K, Yoshikai Y,    Nomoto K. Radioresistant CD4-CD8-intrathymic T cell precursors    differentiate into mature CD4 CD8− and CD4-CD8+ T cells. Development    of ‘radioresistant’ CD4-CD8-intrathymic T cell precursors. Thymus.    1990:15:65-78.-   65. Jones R J, Wagner J E, Celano P, Zicha M S, Sharkis S J.    Separation of pluripotent haematopoietic stem cells from spleen    colony-forming cells. Nature. 1990; 347:188-189.-   66. Seung Woo Lee, Yunji Park, Jae Kwang Yoo, So Young Choi, and    Young Chul Sung. Inhibition of TCR-Induced CD8 T Cell Death by    IL-12: Regulation of Fas Ligand and Cellular FLIP Expression and    Caspase Activation by IL-12. J Immunol. 2003; 170:2456-2460.-   67. Medical College Of Georgia. “Breast Cancer Uses Growth Factors    To Lure Stem Cells.” ScienceDaily, 9 Jun. 2005. Web. Retrieved 29    Jun. 2011.-   68. Kryczek I, Grybos M, Karabon L, Klimczak A, Lange A. IL-6    production in ovarian carcinoma is associated with histiotype and    biological characteristics of the tumour and influences local    immunity. Br J Cancer. 2000; 82:621-628.-   69. Freedman R S, Deavers M, Liu J, Wang E. Peritoneal    inflammation—A microenvironment for Epithelial Ovarian Cancer (EOC).    J Transl Med. 2004; 25:23.-   70. Jackson J D, Yan Y, Brunda M J, Kelsey L S, Talmadge J E.    Interleukin-12 enhances peripheral hematopoiesis in vivo. Blood.    1995; 85:2371-2376.-   71. Zhao Y, Zhan Y, Burke K A, Anderson W F. Soluble factor(s) from    bone marrow cells can rescue lethally irradiated mice by protecting    endogenous hematopoietic stem cells. Exp. Hematol. 2005; 33:428-434.-   72. Epstein F H. Cutaneous wound healing. N Engl J Med. 1999;    34:738-746.-   73. Engelhardt E, Toksoy A, Goebeler M, Debus S, Brocker E B,    Gillitzer R. Chemokines II-8, GROa, MCP-1, IP-10, and MIG are    sequentially and differentially expressed during phase specific    infiltration of leukocyte subsets in human wound healing. Amer. J    Pathol. 1998; 153:1849-1860.-   74. Gillitzer R, Goebeler M. Chemokines in cutaneous wound healing.    J Leuk. Biol. 2001; 69:513-521.-   75. Wu C, Warrier R R, Wang X, Presky D H, Gately M K. Regulation of    interleukin-12 receptor beta1 chain expression and interleukin-12    binding by human peripheral blood mononuclear cells. Eur J Immunol    1997; 27:147-54.-   76. Szabo S J, Dighe A S, Gubler U, Murphy K M. Regulation of the    interleukin (IL)-12R β2 subunit expression in developing T helper    (Th1) and Th2 cells. J Exp Med 1997; 185: 817-24.-   77. Rogge L, Barberis-Maino L, Biffi M, Passini N, Presky D H,    Gubler U, Sinigaglia F. Selective expression of an interleukin-12    receptor component by human T helper 1 cells. J Exp Med 1997;    185:825-31.-   78. Venezia T A, Merchant A A, Ramos C A, Whitehouse N L, Young A S,    Shaw C A, Goodell M A. Molecular signatures of proliferation and    quiescence in hematopoietic stem cells. PLoS Biol. 2004 October;    2(10):e301. Epub 2004 Sep. 28.-   79. Ivanova N B, Dimos J T, Schaniel C, Hackney J A, Moore K A,    Leminchka I R. A stem cell molecular signature. Science. 2002;    298:601-604.-   80. The Lineage Cell Depletion Kit is a magnetic labeling system for    the depletion of mature hematopoietic cells, such as T cells, B    cells, monocytes/macrophages, granulocytes and erythrocytes and    their committed precursors. All the secondary antibodies for this    system are goat anti-rat IgG conjugated with MicroBeads (Miltenyi    Biotec GmbH, Auburn, Calif.)-   81. Liddle R A, Misukonis M A, Pacy L, Balber A E. Cholecystokinin    cells purified by fluorescence-activated cell sorting respond to    monitor peptide with an increase in intracellular calcium. Proc Natl    Acad Sci USA. 1992; 89:5147-51.-   82. Jordan C T, Astle C M, Zawadzki J, Mackarehtschian K, Lemischka    I R, Harrison D E. Long term repopulating abilities of enriched    fetal liver stem cells measured by competitive repopulation. Exp    Hematol. 1995; 23:1011-1015.-   83. de Wynter E, Ploemacher R E. Assays for the assessment of human    hematopoietic stem cells. J Biol Regul Homeost Agents 2001;    15:23-27.-   84. Zhang C C, Lodish H F. Murine hematopoietic stem cells change    their surface phenotype during ex vivo expansion. Blood. 2005;    105.4314-4320.-   85. Torok-Storb B, Iwata M, Graf L, Gianotti J, Horton H, Byrne M C.    Dissecting the marrow microenvironment. Ann NY Acad Sci. 1999;    0;872:164-70.-   86. Shih C C, Hu M C T, Hu J, Weng Y, Yazaki P J, Medeiros J, Forman    S J. A secreted and LIF-mediated stromal cell-derived activity that    promotes ex vivo expansion of human hematopoietic stem cells. Blood.    2000; 95:1957-1966.-   87. Afkarian M, Sedy J R, Yang J. Jacobson N G, Cereb N, Yang S Y,    Murphy T L, Murphy K M. T-bet is a Stat-1-induced regulator of    IL-12R expression in naïve CD4⁺ T cells. Nature Immunol. 2003;    3:549-557.-   88. DeMeyer E S, Baar J. Dendritic Cells: The Sentry Cells of the    Immune System. 2001. Monograph. Oncology Education Services.-   89. Ozato K, Tsujimura H, Tamura T. Toll-like receptor signaling and    regulation of cytokine gene expression in the immune system.    Biotechniques 2002; 70, 72 passim supp 66-68.-   90. Attar E C, Scadden D T. Regulation of hematopoietic stem cell    growth. Leukemia 2004; 18:1760-68.-   91. Wang Q, Zhang W, Ding G, Sun L, Chen G, Ciao X. Dendritic cells    support hematopoiesis of bone marrow cells. Transplantation 2001;    72:891-899.-   92. Jacobsen S E W, Veiby O P, Smeland E B. Cytotoxic lymphocyte    maturation factor (interleukin 12) is a synergistic factor for    hematopoietic stem cells. J. Exp. Med 1993; 178:413-418.-   93. Bystrykh L, Weersing E, Dontje B, Sutton S, Pletcher M T,    Wiltshire T, Su A I, Vellenga E, Wang J, Manly K F, Lu L, Chesler E    J, Alberts R, Jansen R C, Williams R W, Cooke M P, de Haan G.    Uncovering regulatory pathways that affect hematopoietic stem cell    function using ‘genetical genomics’. Nature Genetics 2005;    37:225-232-   94. Zou W. Immunosuppressive networks in the tumour environment and    their therapeutic relevance. Nature Review (Cancer) 2005; Sep.    14-25.-   95. Hideo E, Nakauchi H. Non-side population hematopoietic stem    cells in mouse bone marrow. Blood, first ed.; prepublished online    Jun. 27, 2006.-   96. Paterson H M, Murphy T J, Purcell E J, Shelley O, Kriynovich    S J. Lien E, Mannick J A, Lederer J A. Injury primes the innate    immune system for enhanced toll-like receptor activity. Immunol    2003; 171:1473-1483.-   97. U.S. Pat. No. 7,939,058 entitled “Uses of IL-12 in    Hematopoiesis.”

1-24. (canceled)
 25. A method for repairing or activating cells in asubject comprising: (a) isolating a cell population that expresses theIL-12 receptor; (b) exposing the cell population to exogenous IL-12ligand; and (c) administering the cell population to a subject.
 26. Themethod of claim 25, wherein the cell population is isolated using anantibody that binds the beta 2 subunit of the IL-12 receptor.
 27. Themethod of claim 25, further comprising administering exogenous IL-12ligand to the subject following administration of the cell population.28. The method of claim 25, wherein the subject has diabetes.
 29. Themethod of claim 25, wherein the subject has one or more blood cellcounts that are below normal range due to the effects of radiationtherapy or chemotherapy.
 30. The method of claim 25, wherein the cellpopulation is administered to an organ selected from the groupconsisting of kidney, liver, lung, spleen, pancreas, and cardiac tissue.31-35. (canceled)
 36. A method of regenerating tissue of a subject invivo comprising: administering exogenous IL-12 ligand to a subject;wherein the exogenous IL-12 ligand binds to the IL-12 receptor on apopulation of cells in the tissue to yield an increase in the numberIL-12 receptor positive stem cells.
 37. The method of claim 36, whereina route of exogenous IL-12 ligand administration is selected from thegroup consisting of epidural, peridural, intracerebral, intrathecal,intracerebroventricular, enteral, subcutaneous, intravenous,intraarterial, intramuscular, intrauterine, and intraperitoneal.
 38. Themethod of claim 36, wherein the method is for regenerating kidney tissueof a subject in vivo and wherein the exogenous IL-12 ligand binds to theIL-12 receptor on a population of kidney cells to yield an increase inthe number IL-12 receptor positive stem cells. 39-41. (canceled)
 42. Themethod of claim 36, wherein the method is for regenerating intestinaltissue of a subject in vivo and wherein the exogenous IL-12 ligand bindsto the IL-12 receptor on a population of intestinal cells to yield anincrease in the number IL-12 receptor positive stem cells. 43-44.(canceled)
 45. The method of claim 25, wherein the subject has cancer.46. The method of claim 45, wherein the cell population is administeredby intravenous infusion.
 47. The method of claim 36, wherein the methodis for regenerating neuronal tissue of a subject in vivo and wherein theexogenous IL-12 ligand binds to the IL-12 receptor on a population ofneuronal cells to yield an increase in the number IL-12 receptorpositive stem cells.
 48. The method of claim 25, wherein the exogenousIL-12 ligand comprises at least one IL-12 ligand selected from the groupconsisting of IL-12 heterodimer ligand, IL-12 homodimer ligand, IL-12monomer ligand, a p35 subunit of an IL-12 heterodimer ligand, and a p40subunit of an IL-12 heterodimer ligand.
 49. The method of claim 48,wherein the IL-12 ligand is glycosylated.
 50. The method of claim 36,wherein the exogenous IL-12 ligand comprises at least one IL-12 ligandselected from the group consisting of IL-12 heterodimer ligand, IL-12homodimer ligand, IL-12 monomer ligand, a p35 subunit of an IL-12heterodimer ligand, and a p40 subunit of an IL-12 heterodimer ligand.51. The method of claim 50, wherein the IL-12 ligand is glycosylated.52. The method of claim 27, wherein the exogenous IL-12 ligand isadministered at a dose of about 100 ng/kg up to about 500 ng/kg.
 53. Themethod of claim 52, wherein the exogenous IL-12 ligand is administeredat a dose of about 397.4 ng/kg or less.
 54. The method of claim 52,wherein the exogenous IL-12 ligand is administered at a dose of about250 ng/kg or less.
 55. The method of claim 36, wherein the exogenousIL-12 ligand is administered at a dose of about 100 ng/kg up to about500 ng/kg.
 56. The method of claim 55, wherein the exogenous IL-12ligand is administered at a dose of about 397.4 ng/kg or less.
 57. Themethod of claim 55, wherein the exogenous IL-12 ligand is administeredat a dose of about 250 ng/kg or less.